Cooling Adjunct For Medications To Treat Disorders In The Nasal Cavity

ABSTRACT

The present discovery pertains generally to the field of therapeutic compounds. More specifically the present discovery pertains to certain di-alkyl-phosphinoyl-alkanes as described herein, DIPA-1-8 and DIPA-1-9, and 2-6 and 2-7 that are collectively referred to herein as “DAPA compounds”, that are useful in the treatment of disorders (e.g., diseases) including: sensory discomfort (e.g., caused by inflammation, irritation, itch, or pain) in the nasal cavity. The applicant has found that localized delivery of DAPA compounds in combination with an intranasal steroid or an intranasal antihistamine immediately relieves nasal discomfort and enhances patient adherence to the use of the nasal medications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 14/545,014filed Mar. 16, 2015.

Inventor: Edward Tak Wei

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains generally to the formulation oftherapeutic compounds for treatment of disorders of the nasal cavity.More specifically the present invention pertains to use of certaincooling agents that are useful in the relief of sensory discomfortoriginating from the nasal cavity and the incorporation of such coolingagents with another active ingredient in pharmaceutical compositions,and the use of these combinations for topical delivery of such compoundsand compositions to the membranes of the nasal cavity.

Description of Related Art

Chemical sensory/cooling agents are molecules that can mimic thesensations of heat abstraction without a change in tissue temperatures.The exact sensations produced by chemicals depend on the selection ofthe active ingredient and the site and method of delivery. The term“chemical cooling agent” can be ambiguous because, for example,chemicals such as ethyl chloride as a gas, ethanol as a liquid, liquidnitrogen, or carbon dioxide as a solid, applied to the skin can evokeheat abstraction sensations by reducing tissue temperatures. In thisapplication, chemical cooling agents will refer only to agents thatelicit sensations of heat abstraction without a lowering of tissuetemperatures.

The lining of the nasal membranes, called respiratory epithelium, isonly one cell layer thick. At the base of this single layer are sensorynerve endings that are connected to the trigeminal sensory nerve. Thesenerve endings are highly sensitive to temperature, irritants, humidity,and osmotic pressure. One manifestation of nasal discomfort andinflammation is nasal stuffiness and congestion, and a sense of loss ofpatency and obstructed airflow. This condition can have many causes, themost common being “rhinitis”, a technical term meaning the condition ofinflammation of the membranes lining the nose. Rhinitis, especiallyallergic rhinitis, is characterized by nasal congestion, rhinorrhea(“runny nose”), sneezing, itching of the nose and/or postnasal drainage.A common form of rhinitis is seasonal allergic rhinitis which is causedby seasonal aeroallergens such as pollens and molds [Bousquet et al.Allergic Rhinitis and its Impact on Asthma Eur. J. Allergy Clin.Immunol. 63, 8-160 (2008); Seidman, M. D. et al. Clinical PracticeGuideline: Allergic Rhinitis. Otolaryngol. Head Neck Surg. 152, S1-S43(2015)]. Perennial allergic rhinitis is caused by perennialenvironmental aeroallergens such as dust mites, molds, animal allergens,or occupational allergens. Rhinitis can also be caused by foodallergies. Some individuals, without evidence of allergic sensitization,will have rhinitis in reaction to nonspecific irritant stimuli such ascold dry air, perfumes, paint fumes, and cigarette smoke. This conditionis called vasomotor rhinitis. Severe rhinitis may result from injury tothe nasal membranes such as occurs after smoke inhalation, sinusitis, orafter nasal surgery.

Rhinitis is also caused by the common cold virus. Initially, viralrhinitis is characterized by clear, watery rhinorrhea that isaccompanied by sneezing and nasal obstruction. Edema of the nasal mucosaproduces occlusion of the sinus ostia, with resulting facial pain, or ofthe Eustachian tube, with resulting ear fullness. Responsible virusesinclude rhinoviruses, respiratory syncytial virus, parainfluenza,influenza and adenoviruses. Fever may accompany viral rhinitis,especially if there is bacterial superinfection by streptococcalorganisms.

The sinuses drain into the nasal cavity. Rhinosinusitis is inflammationof the mucosa of the nasal sinuses together with the nasal membranes.This condition is a major cause of breathing discomfort because it isaccompanied by prolonged mucopurulent nasal discharge, facial pain andpressure, olfactory disturbance, and post-nasal drainage with cough. Inrhinosinusitis, the causes of discomfort may infectious organisms suchas bacteria and fungi. Hence, topical antibiotics, including anti-fungalagents may be applied, which have irritant properties of their own.Saline is also used for the irrigation of the sinuses. The large volumeof liquid forced through the nasal cavity may cause mechanicaldiscomfort.

Neoplastic diseases of the nasal cavity can also occur, including nasalcarcinoma and metastases from the nasopharyngeal cavity. In thesesituations, cancer chemotherapeutic agents or radiation will causerhinitis and sensory discomfort.

Rhinitis is a common symptom. There are approximately 60 millionindividuals with rhinitis each in the USA and in Europe, and about 30million in Japan. The prevalence of allergic rhinitis, a subset ofrhinitis, is estimated to be up to 20+% of the general population. Thecommon cold also causes rhinitis and in the USA each person has one ortwo bouts of per year. The economic burdens of rhinitis associated withallocation of health resources, from loss of work days, and from absenceat schools are significant [Stewart, M. et al. Epidemiology and burdenof nasal congestion. Int. J. Gen. Med. 3, 37-45 (2010)].

Pharmacological management of some forms of rhinitis, especiallyallergic rhinitis, is a well-developed science. Effective topicalmedications for allergic rhinitis are the intra-nasally administeredglucocorticosteroids [intranasal steroids], intranasal antihistamines,and sympathomimetic decongestants. Ancillary medications are the mastcell stabilizer, called sodium cromolyn, and ipratropium which reducesrhinorrhea but does not affect sneezing or itch. Other treatmentsinclude hypertonic saline or isotonic saline for nasal cavityirrigation. Sprays, nose drops, solutions, and gels are used for topicaltreatments of the nasal membranes to deliver the active ingredients andare familiar items of patient use for example, manual pump-operatedmetered atomizers (e.g. Flonase® and Nasacort®). Examples of leadingcorticosteroids used for allergic rhinitis are beclomethasonedipropionate, triamcinolone acetonide, budesonide, fluticasonepropionate, mometasone furoate, ciclesonide, and fluticasone furoate.Examples of topical antihistamines are olopatadine and azelastinehydrochloride. The intranasal steroids and antihistamines reduce nasalmembrane inflammation and the symptoms and signs of allergic rhinitis.Intranasal steroids are not effective for relieving the discomforts ofinfectious [e.g. viral] rhinitis, and have limited efficacy for therhinitis caused by rhinosinusitis. Intranasal steroids andantihistamines are less effective for rhinitis caused by air pollutantswherein irritants directly damage the nasal mucosa.

Menthol, camphor and eucalyptus oil have been used since ancient timesas remedies for nasal irritation and for refreshment of nasalsensations. These compounds may briefly provide cooling sensations inthe nasal passages but are not effective for rhinitis. In fact thesesubstances exacerbate nasal congestion and obstruction, especially inthe late and delayed stages of rhinitis. In the laboratory, menthol isan irritant when instilled into the nasal passages of humans [Alenmyr,L. et al. TRPV1 and TRPA1 stimulation induces MUC5B secretion in thehuman nasal airway in vivo. Clin. Physiol. Funct. Imaging 31, 435-444(2011)]. Menthol vapor delivered onto the membranes of the nasopharynxvia orthograde or retrograde airflow [in the form of a menthol lozenge]has pungency and a cooling effect which briefly relieves nasaldiscomfort. The pungency of menthol may stimulate vasoconstriction ofthe nasal blood vessels and this contributes to a brief decongestantaction.

The sympathomimetic vasoconstrictors (decongestants) reduce nasal bloodflow and symptoms of congestion, but these compounds have a number ofadverse side-effects, including rebound hyperemia (rhinitismedicamentosa). The recommended dosing schedule is not to exceed oneweek of use, and preferably not more than three days.

It is a common experience that breathing cool air, for example at theseaside, will enhance the sense of fresh airflow in the nose. Thiseffect has been demonstrated in the laboratory where subjects report agreater sense of nasal patency with lower nasal septum temperatures[Willatt et al. The role of the temperature of the nasal lining in thesensation of nasal patency. Clin. Otolaryngol. Allied Sci. 21, 519-523(1996)]. Wei [U.S. Pat. No. 6,933,301. Aug. 23, 2005] proposed that acooling agent, called icilin, administered into the nasal cavity may beuseful for the relief of the symptoms of rhinitis, but this idea was notcommercialized because of technical difficulties in formulating icilinfor delivery into the nasal cavity.

Although physicians recommend intranasal steroids as the firstmedication of choice for the treatment of allergic rhinitis [Seidman etal., vide supra], patient adherence to effective use of intranasalsteroids is variable [36% to 64%] In a survey conducted in 2012, only ⅓of the patients with seasonal allergies used intranasal steroids fortreatment [Fromer et al. J Family Practice Insights on allergic rhinitisfrom the patient perspective. 61: S16-22, 2012]. In controlled trials,however, the intranasal steroids give the most benefit to patients and,with the advent of over-the-counter approved intranasal steroids such asNasacort®, Flonase®, and Rhinocort®, a relatively inexpensive andeffective medication exists for the treatment of allergic rhinitis.Treatment of allergic rhinitis, and other forms of rhinitis, can beimproved if there is a procedure to enhance patient acceptance of andadherence to an intranasally administered medication. Among the reasonsfor non-adherence are forgetfulness, the fear of side effects ofintranasal steroids, bad taste or smell from the spray, alternativedrugs (e.g. oral antihistamines), and the lack of immediate ofsymptomatic relief [Marple et al. Keys to successful management ofpatients with allergic rhinitis: focus on patient confidence,compliance, and satisfaction., Otolaryngol. Head. Neck Surg. 136,S107-24 (2007)].

BRIEF SUMMARY OF THE INVENTION

The preferred embodiments of this discovery (Formula 1 compounds) in anaqueous solution of 1 to 10 mg/mL and delivered at a volume of ˜0.1 mLper nostril, will provide immediate (<2 min) relief of nasal irritationand congestion. The refreshing clearing of the nasal passages is feltupon spraying or the instillation of nose drops. This feature ofimmediate cooling and relief of nasal congestion will increase patientadherence/compliance to the use of the intranasal medication.

In one embodiment of the present invention, a combination composition isprovided that comprises a cooling agent such as a1-[Dialkyl-phosphinoyl]-alkane compound of Formula 1:

(O═)PR₁R₂R₃

-   -   wherein each of R₁, R₂, is either isopropyl or sec-butyl and R₃        is a linear alkyl group of 6 to 9 carbons,    -   and a topical medication for the treatment of nasal cavity        disorders, such as an intranasal steroid, a intranasal        anthistamine, a mast cell stabilizer, a sympathomimetic        decongestant, a muscarinic antagonist, an antibacterial agent,        an antifungal agent, or a saline solution for nasal irrigation.

Compounds of Formula 1 are an adjunct formulated into the combinationcomposition. The composition is usefully delivered in a therapeuticallyeffective amount as a solution onto the membranes of the nasal cavity,preferably by means of a spray, nose drops, or gel. A preferredembodiment of the compound of Formula 1 is represented by1-[Diisopropyl-phosphinoyl]-octane [DIPA-1-8] or by1-[Diisopropyl-phosphinoyl]-nonane [DIPA-1-9].

The preferred embodiments of the adjunct, DIPA-1-8 and DIPA-1-9 wereselected because they impart an immediate, robust, and refreshingsensation to the nasal mucosa, without an effect on the nostril skin orpungency, and, at selected concentrations ranging from 0.01 to 1%,elicit a long-lasting enhancement of nasal refreshment. Uponinstillation into the nostrils, the effects have immediate onset and arepenetrating and refreshingly cool. Practice of this invention providestherapeutic medications that have improved acceptance and adherence bypatients for the treatment of nasal cavity discomforts. Particularly forsuch sensations of nasal obstruction and nasal congestion caused, forexample, by allergic rhinitis or rhinosinusitis.

An adjunct used in a medication is an additional substance or treatmentused for increasing the efficacy or safety of the primary substance. TheDAPA compounds relieve sensory discomfort in the nasal cavity. It isproposed that they be used as adjuncts with other pharmaceuticals forthe nasal cavity site.

An adjunct such as DIPA-1-9 will facilitate “apparent” efficacy ofanother primary ingredient, and thereby improve patient satisfaction,compliance, and adherence to a dosage schedule. For example, DIPA-1-9may be combined with an intranasal anti-inflammatory steroid such astriamcinolone acetonide in a formulation for rhinitis. The preparationmay be more desirable than the anti-inflammatory steroid alone, whichtakes longer to act. Anti-inflammatory steroids used for intranasalapplications include beclomethasone dipropionate, triamcinoloneacetonide, budesonide, fluticasone propionate, mometasone furoate,ciclesonide, and fluticasone furoate. Other examples of intranasal drugswhich may be combined with the DAPA compounds of this discovery include:antihistamines for intranasal applications such as olopatadine,azelastine, and levocabastine; sympathomimetic amine vasoconstrictorssuch as phenylephrine hydrochloride, oxymetazoline, naphazoline, andother imidazoline receptor agonists; ipratropium bromide which is anantimuscarinic agent used especially for rhinorrhea; sodium cromolynwhich a mast cell stabilizer; and ketorolac, a non-steroidalanti-inflammatory agent; amphotericin B which is a anti-fungal agent;surfactants; and hypertonic and isotonic salne—all of which are used fortopical medication of nasal-sinus membranes.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. is a graph of TRPM8 potency of Diisopropyl-phosphinoyl-alkaneand Di-sec-butyl-phosphinoyl alkane analogs in the in vitro TRPM8 assay.Units are in comparison to I-menthol potency in the same assay. Thenumbers on the abscissa represent the number of carbons in one of then-alkyl side-chain: that is, the 4-5-6-7-8-9 represents the butyl,pentyl, hexyl, heptyl, octyl, and nonyl group, respectively.

FIG. 2. is a graph showing the lack of agonist activity of DIPA-1-8 incells transfected with TRPV1 or TRPA1 plasmids. The positive controlscapsaicin and mustard oil for TRPV1 and TRPA1 are active, respectively,but DIPA-1-8 is not active in these TRPV1 or TRPA1 transfected cells.The ordinate is given in Relative Fluorescence Units % of maximum, whichmeasure calcium entry into the transfected cells and the abscissa is thelogarithm of the concentration of the test compound.

FIG. 3. is a graph showing the absence of tachyphylaxis to the shakingeffects of repeated injections of DIPA-1-7, 2 mg/kg intravenous, at 15min intervals. It can be seen that the shaking intensity of the 3^(rd)and 4^(th) trials were not diminished by the previous injections.

DETAILED DESCRIPTION OF THE INVENTION

The nares, the pair of openings below the tip of the nose, are theentrances to the respiratory tract. The nasal passages serve as aconduit for inspired and expired air. When these passages are congestedor obstructed, the condition is perceived as uncomfortable. The nasalcavity bony surfaces, including the sinuses, are lined by tissue calledmucosa. This mucosa contains blood vessels, nerves, and small glandsthat secrete mucus and fluids into the nasal cavity. The respiratoryepithelium which covers most of the nasal mucosa is only one cell layerthick and ciliated. The nasal mucosa is richly supplied by sensorynerves that detect pain, temperature, pressure and odor, and by motornerves that regulate secretions and blood flow. The nasal mucosahumidifies and warms the inspired air, hence it receives a large bloodflow and the cells maintain a high degree of metabolic activity.Inflammation of the nasal mucosa caused by allergy, infections, injury,or irritants and the like, will stimulate the mucosa to secrete fluids,to swell, and to obstruct. When the nasal membranes increase in volume,the area available through which air can pass is diminished, andtherefore one experiences a sense of “stuffiness”, resistance toinspiration, and a feeling of nasal obstruction and loss of patency. Thenose can also become itchy and “runny” (rhinorrhea) and, as the fluidsand discharges accumulate, the result is a feeling of congestion anddiscomfort.

The following descriptions give an over-view of some quantitativedimensions of nasal function. The normal air intake is about 10,000liters per day and nasal secretions contribute about 30 ml of fluids tohumidify each 1000 liters [300 mL per day is about the volume of a canof soda]. The relative humidity of dry air inhaled via the nose is about60% when it goes past the nose, but it is only about 5% when the air isbreathed through the mouth. The relative humidity of air in the bronchiis 100% at body temperature and this humidification, contributed byblood flow through the mucosa, is required to maintain ciliary activityand prevent epithelial changes in the bronchial mucosa. Desiccation ofthe bronchial surface for more than 2 to 3 hours can cause mucosalchanges that result in thickening of secretions, irritation, andincreased susceptibility to infection.

Abbreviations and Terminology

Adherence or Compliance—The two terms, used interchangeably, describethe degree to which a patient correctly follows professional advice onuse of medication. Barriers to compliance include the clarity ofinstructions on proper use of the medication, poor patient understandingof treatment benefits, fear of and actual occurrence of side effects,the costs of the medicine, and the degree of understanding betweenpatient and adviser. Frequently, a patient will listen to the advice offriends and use alternative medications such as vitamin supplements orherbal preparations if the prescribed medication does not work quickly.Adherence can be improved if there is immediate relief, by simplifyingmedication packaging, by providing effective reminders, by improvingpatient education, and/or by limiting the number of medicationsprescribed simultaneously. In this discovery, a pharmacological “boost”to a topical nasal cavity medication is provided by incorporating acooling agent into the formulation. The goal is to give patientimmediate relief of nasal congestion. The patient, by experiencingrefreshed breathing may then adhere better to the treatment regimen, andlet other drug actions, e.g. anti-inflammatory and anti-histaminergicbecome manifest. The term “adherence” now is preferred to “compliance”(by the WHO, American Pharmacists Association, and the NIH) becauseadherence implies the patient has greater freedom in cooperating withthe health professional and is not just following didactic instructions.Both terms are accepted.

DAPA and DIPA compounds, DAPA and DIPA is the abbreviations for1-[Dialkyl-phosphinoyl]-alkane and 1-[Diisopropyl-phosphinoyl]-alkane,respectively. 1-[Di-sec-butyl-phosphinoyl]-alkanes are also described inthis application. An alternative name is 1-dialkylphosphorylalkanes. Thethird alkyl group in the molecule may be described by a number: hence,4, 5, 6, 7, 8, 9, and 10 correspond to the butyl, pentyl, hexyl, heptyl,octyl, nonyl, and decanyl side chain, respectively. These alkanes arelinear or “normal [n]” in configuration, with the phosphinoyl groupattached to the primary, or “1-” position, of the carbon chain in thethird sidechain. The syntheses of DIPA-5 to 7 and DIPA-9 to 10 were notpreviously reported but Siddall et al. [Simplified preparation of sometrisubstituted phosphine oxides. J. Chemical Engineering Data 10:303-305, 1965] reported the synthesis of DIPA 1-8 in 1965. Thebiological activities of the DIPA compounds applied to any organismicsurfaces [e.g nasal cavity] have not been reported prior to thisdiscovery.

Nasal patency—is the subjective sensation of openness and smooth airflowin the nasal passages when breathing. Loss of patency may be reported as“nasal stuffiness” or “nasal blockage” and causes discomfort, and thesubject may use mouth breathing which is undesirable because itdesiccates the airway surfaces. Simple compression of the alar creasewith the thumb and fore finger will cause the sense of impededbreathing. Nasal congestion implies excess fluids in the nasal passages.The term “nasal obstruction” is used more often when there is aphysical, for example anatomical or structural, hindrance to airflow.Rhinitis, the inflammation of the membranes in the nasal cavity, is mostoften associated with nasal congestion and nasal obstruction. In the“empty nose syndrome” there are severe breathing discomforts, includingloss of the sense of patency, but the symptoms can occur withoutrhinitis or physical evidence of change in airflow or gas exchange[Sozansky, J. Pathophysiology of empty nose syndrome. Laryngoscope 125,70-74 (2015)].

Topical versus Systemic Administration—in the context of this discovery,topical administration is intranasal delivery of the drug ingredientonto the membranes of the nasal cavity directly via a spray, nose drops,or gel. Systemic administration is delivery of the drug to nasalmembranes after passage into and distribution via the blood stream.Topical administration is safer than systemic in principle because theeffective dose is much lower than the systemic effective dose due toless dilution in the body mass. To be effective, the topical activeingredient must not locally irritate the one-cell layer of the nasalepithelium.

Total Nasal Symptom Score (TNSS)—is a method, using a standardizedquestionnaire, to evaluate symptoms of nasal function in patients withallergic rhinitis. Scores are rated individually for: nasal congestion,runny nose, nasal itching, sneezing and difficult sleep, on a scale ofnone (0), mild (1), moderate (2), and severe (3) and the sum of thescores is the TNSS (min=0, max=15).

Cooling Agents and Nasal Cavity Physiology

Nasal cavity volume is not fixed by anatomy but fluctuates with theamount of blood in the venous capacitance vessels of the nose. In thenasal cycle, the side of the nose that is increased in volume is filledwith the blood that is used to heat the incoming air. The blood supplyto the nasal mucosa comes from five arteries. Both the internal andexternal carotid circulations contribute to the arterial supply of thenasal cavity. The anterior and posterior ethmoid arteries, branches ofthe ophthalmic artery, enter the nose after passing through the orbitand the lamina papyracea. The sphenopalatine artery, a terminal branchof the external carotid artery, enters the nose through the posteriorlateral inferior wall. Additional blood supply comes from the greaterpalantine artery and the superior labial artery. Four of these vessels[the posterior ethmoid artery does not participate] anastomose and forma plexus of fenestrated capillaries some of which face the respiratorysurface and are the major source of fluids and heat for humidifying andwarming the air in the nasal cavity. Intranasal trigeminal fibers aredistributed throughout the nasal cavity and are described asintraepithelial free nerve endings arising from A6 and C fibers of thenasopalatine and ethmoid branches of the trigeminal nerve. This neuralnetwork controls blood flow and secretions [Sahin-Yimaz A et al. Anatomyand physiology of the upper airway. Proc. Am. Thoracic Soc. 8:31-39,2011].

It has been known for some time that an individual's perception of nasalpatency [for example, on a visual analog scale of 0 to 10 with 0 forbeing clear and 10 for being blocked] is not readily correlated toobjective measurements of nasal airflow or nasal cavity volume [Zhao, K.et al. Regional peak mucosal cooling predicts the perception of nasalpatency. Laryngoscope 124, 589-595 (2014)]. Thus, physicians have beenpuzzled by the lack of consistent correlation between a patient'ssubjective complaints of congestion/blockage versus objectivemeasurements, such as rhinomanometry for nasal airflow resistance,acoustic rhinometry for nasal cavity volume, and endoscopic examinationof the nasal cavity. Without objective measurements of nasal functionthat relate to symptoms, medical treatment must rely the patient'sopinions of patency for treatment success and this can lead to confusingoutcomes, especially if surgery is contemplated.

Breathing cool air increases the sense of nasal patency. This is a factof common experience, for example, breathing at the seaside. It has beenshown in the laboratory when the inspired air temperature measured atthe septum is kept at 25 to 35° C. there is a greater sense of patencyat the lower temperature [Willatt et al., vide supra]. However, it isalso well-known that cold and frigid air will evoke a “runny nose” orrhinorrhea, an event mediated by cholinergic nerves on serous glands ofthe nasal epithelium [Ostberg et al. Cold air induced rhinorrhea andhigh-dose ipratropium Arch. Otolaryngol. Head and Neck Surg. 113,160-162 (1996)]. This condition has also been called a “skier's nose”and is quite common. Thus, cooling of the nasal cavity may increasesecretions and exacerbate congestion. The negative effect of cooling oncongestion is further shown by the fact that lowering the bodytemperature by immersion in cold water will cause vasodilation andincreased nasal mucosal blood flow and hot water causes the nasalarteries to constrict [Lundqvist et al. Nasal reaction to changes inwhole body temperature. Acta Otolaryngol. 113, 783-786 (1993)]. Thus,one cannot predict, ipso facto, that colder air temperatures willrelieve congestion because cold causes rhinorrhea and increases nasalcavity volume.

The nasal afferents for detection of temperature are located in branchesof the trigeminal nerve. The most likely receptor on the nerve endingsmediating detection of coolness is the voltage-dependent cation channelcalled TRPM8, although a Grueneberg neuron receptor called CNGA3 mayalso participate [Mamasuew et al. The cyclic nucleotide-gated ionchannel CNGA3 contributes to coolness-induced responses of Gruenebergganglion neurons. Cell. Mol. Life Sci. 67, 1859-1869 (2010)]. Keh et al.[The menthol and cold sensation receptor TRPM8 in normal human nasalmucosa and rhinitis. Rhinology 49, 453-7 (2011)] have detected TRPM8immunoreactivity in human nasal mucosa, closely associated with nervefibers and blood vessels. The immunoreactive TRPM8 proteins in the nasalmucosa were not increased in patients with rhinitis. Keh et al. [2011]suggested that TRPM8 antagonists might have value in rhinitis. I proposehere an opposite view; namely, TRPM8 agonists have beneficial effects inthe nasal discomfort caused by rhinitis. In science there is often timesconfusion when one group says that the agonist will work, and anothergroup advocates the antagonist. The data in this application clearlyfavor the agonists and not the antagonists.

The nerves and blood vessels of the nasal mucosa regulate thetemperature and the humidification of air. The nerve endings participatein the inflammatory response by releasing transmitters, such ashistamine, acetylcholine, CGRP, and substance P, that together withcells of the immune system control blood flow secretions. The sensorsand effectors are highly concentrated in the anterior portion of thenose, at the anatomical juncture for the detection of the airtemperature entering the nostrils. At first, one might hypothesize thatcooling agents administered intranasally will inhibit the sensations ofcongestion by masking the signals of fullness and distension thataccompanies nasal congestion. But this view is not correct becauseinhalation of menthol or inhalation of very cold air have limitedbeneficial effects on nasal stuffiness in clinical situations andmenthol and cold stimulate rhinorrhea. Decreasing body temperatureincreases nasal mucosal blood flow [presumably to warm the incomingair].

Hypothesis on Mechanism of Drug Action

Without being bound by theory, I propose here a mechanism of drug actionthat might explain the findings in this invention. To go over the mainobservations:

-   -   the delivery of microgram amounts of the DAPA compounds of this        discovery to the anterior portion of the nasal cavity produces        refreshing coolness and promotes an immediate sense of nasal        patency.    -   the benefit with one application of a DAPA compound for rhinitis        is prolonged, lasting for 3+hr in some subjects    -   DIPA analogs that produce intense cold, e,g. DIPA-1-7, are not        as effective as compounds that produce mild cooling sensations,        e.g. DIPA-1-9    -   the selection of the active ingredient is based on the avoidance        of excess cold sensation on nostril skin, and an optimal mild        cooling of the nasal mucosa: hence the choice of active        ingredient is not based on receptor potency, but on selective        activity on nasal mucosa    -   the onset of drug action to relieve congestion and to reduce        nasal secretions occurs quickly, within minutes after        application of the compound area, and the subjective description        of the drug effect is “Amazing!” or “Miraculous!”.    -   this type of drug action has not been previously described for        the treatment of nasal discomfort and is qualitatively distinct        from the actions of intranasal glucocorticosteroids or        antihistamines    -   it is proposed that this pharmacological action of the preferred        embodiments would be ideal as an adjunct in combination with        other medications that act topically in the nasal cavity

TRPM8 Agonists as Adjuncts for Nasal Cavity Medications.

Standard medications for certain forms of rhinitis (e.g. allergicrhinitis) are the intranasal corticosteroids and intranasalantihistamines. But these drugs do not work for rhinitis caused by thecommon cold or air pollutants. Anticholinergic drugs such as ipratropiumand α-adrenergic sympathomimetic decongestants (e.g. phenyephrine,oxymetazoline, napthoazoline) are used, but these drugs have limitedefficacy. A combination of azelastine and mometasone has been introducedfor allergic rhinitis (Dymista®), but generally combination drugs ofnasal medications are infrequently used.

These criteria were defined as a desirable in an ideal activepharmacological ingredient [API] to be used as an adjunct in combinationwith a topical nasal cavity medications:

-   -   the adjunct API should be easy to formulate with the nasal        medication, e.g. a steroid. Ideally, the active ingredient        should be water soluble at an effective therapeutic        concentration at standard temperature and pressure    -   the adjunct API should be selective for TRPM8 and not active at        other TRP sensory channels such as TRPV1 and TRPA1    -   the adjunct API when delivered should immediately refresh,        provide a sense of cleansing, and increase the sense of nasal        patency to a degree of therapeutic relevance    -   the adjunct API should not over stimulate cold-sensing elements        to cause “stinging cold” or cold rhinorrhea    -   the adjunct API should be chemically stable in solution and not        have odor or irritancy, and have an adequate safety margin    -   the adjunct API should be potent, for example, at 0.1 to 5        mg/mL, and have a duration of drug action that is clinically        meaningful    -   the adjunct API should penetrate tissue barriers to reach        targets in the presence of exudates in the nasal cavity    -   the adjunct API should act in less than 5 min after topical        delivery, thus favoring patient compliance and cooperation

As shown in the studies described below, “ideal” API candidates wereidentified and comprise molecules of Formula 1; in particular, thepreferred embodiment known as DIPA-1-8 and DIPA-1-9.

Pharmaceutical Adjunct and Rationale for Use in Rhinitis Medications

An adjunct is a substance added to a pharmaceutical to increaseefficacy, adherence, or safety of the primary substance. In thisdiscovery, a cooling agent is added to formulations to enhanceadherence/compliance for use of a nasal cavity medication, especially amedication for rhinitis, and more particularly allergic rhinitis. Thepreferred embodiments are DAPA compounds that produce an immediate senseof refreshment to the nasal cavity and relieve nasal congestion andhence increase adherence.

Intranasal steroids, intranasal antihistamines, and decongestants arewidely used in the treatment of allergic rhinitis. Marple et al. [videsupra] notes that there are at least 35 OTC medications and 28prescription medications for allergic rhinitis. In a carefully conductedsurvey of 1200+ adults patients with seasonal allergic rhinitis, Marpleet al. considered the factors for efficient management and adherence inthe drug treatment of allergic rhinitis. Approximately 64% of suchpatients can forget to take their medications and one third considertheir medications to be ineffective. Other factors for poor managementand non-adherence include: dysphoric sedation and dry eyes and mouth inthe first generation oral antihistamines; frequent dosing issues ofsecond generation oral antihistamines and adequate management of plasmalevels of drugs; the topical intranasal antihistamine azelastine has abitter taste and work less well on the inflammatory aspects of rhinitis;intranasal decongestants run the risks of rebound hyperemia when usedfor more than 3 days; antimuscarinic agents work on rhinorrhea but donot seem to affect the sense of congestion, sneezing or itch; and sodiumcromolyn has limited efficacy and slow onset. The intranasalcorticosteroids are the best drug treatment for allergic rhinitis butearlier formulations had odor and affected smell and taste. Side effectssuch as epistaxis, pharyngitis, and nasal irritation were noted and someanalogs had the systemic toxic effects of excess steroids, such asreduction in growth. Package inserts of intranasal steroids also warnedof teratogenic effects in laboratory animals and possible increasedrisks of headaches. Amid such an abundance of confusing information, itis not surprising to a find a high rate of non-adherence.

Marple et al. in their comprehensive survey noted that patients rankedrapid, long-lasting, relief of symptoms as their highest desire in amedication (85 to 88% of the patients ranked these features as beingimportant). Intranasal steroids can act as fast as 30 min but generallytake a day or longer, up to one week, to be fully effective. Otherpriorities were: absence of side effects (e.g. drowsiness), low costs,easy to self-administer, compatibility with other medications, non-habitforming, dosing on an “as needed basis”, “steroid free”, and no aftertaste. An OTC preparation such as Nasacort® fufil virtually all of thedesired qualities in an allergic rhinitis medication, except for being“steroid free” or having an immediate, rapid onset of action.

As shown in the Examples, an adjunct such as the preferred embodimentDIPA-1-9, administered into the nasal cavity will promote a sense ofimmediate clearing and relief of nasal congestion that lasts for hours.Thus, such an adjunct will facilitate “apparent” efficacy of anotherprimary ingredient, and thereby improve patient satisfaction andadherence to a dosage schedule. For example, DIPA-1-9 may be combinedwith an intranasal anti-inflammatory steroid. The preparation may bemore desirable than the anti-inflammatory steroid alone, which takeslonger to act. Anti-inflammatory steroids used for intranasalapplications include such beclomethasone dipropionate, triamcinoloneacetonide, budesonide, fluticasone propionate, mometasone furoate,ciclesonide, and fluticasone furoate. Other examples of intranasal drugswhich may be combined with the DAPA compounds of this discovery include:antihistamines for intranasal applications such as olopatadine,azelastine, and levocabastine; sympathomimetic amine vasoconstrictorssuch as phenylephrine hydrochloride, oxymetazoline, naphazoline, andother imidazoline receptor agonists, antimuscarinic agents such asipratropium, a mast cell stabilizer such as sodium cromolyn andketorolac which is approved for nasal administration. The adjunct DAPAcompound can be also be used for combination medications such Dymista,which is a combination of mometasone furoate and azelastine chlorhydrate(Lulla and Malhotra. U.S. Pat. No. 8,937,057: Combination of azelastineand mometasone for nasal administration). The adjunct assistedmedication may be useful for veterinarian as well as human therapy.

To my knowledge, the proposal to combine a fast-acting cooling agentwith an intranasal steroid or an intranasal antihistamine to enhancepatient acceptance and adherence to the use of nasal medication has notbeen previously described.

Study 1 1-[Dialkyl-phosphinoyl]-alkanes (DAPA)

The [Dialkyl-phosphinoyl]-alkanes [e.g. total number of carbons≦15] aresolvent-like molecules that require only several [1 to 3] steps forsynthesis. They are also known as trialkylphosphine oxides or1-dialkylphophorylalkanes, but the term used here is[Dialkyl-phosphinoyl]-alkane [DAPA]. If two of the alkyl groups areisopropyl, the DAPA is abbreviated to DIPA[Diisopropyl-phosphinoyl-alkane].

Rowsell and Spring [Phosphine oxides having a physiological coolingeffect. U.S. Pat. No. 4,070,496. Jan. 24, 1978], described a range ofphosphine oxides which have a physiological cooling effect on skin andon the mucous membranes of the body, particularly the mouth, throat andgastrointestinal tract [columns 3 and 4 therein]. Ten (10) of thecompounds shown in Rowsell have one isopropyl group. None of thecompounds synthesized by Rowsell and Spring has two isopropyl groups ora n-nonane substituent. Rowsell and Spring considered the use of thetrialkylphopshine oxides as a decongestant in combination withephedrine, but is silent on the use of such compounds with intranasalsteroids or intransal antihistamines, and on the questions of rapidonset of drug action and patient compliance in the use of nasalmedications.

Siddall et al. [Simplified preparation of some trisubstituted phosphineoxides. J. Chemical Engineering Data 10: 303-305, 1965] reported thesynthesis of 1-[Diisopropyl-phosphinoyl]-octane [DIPA-1-8]. No reportson any bioactivity of this or other diisopropyl-phosphinoyl-alkanecompounds have previously been made.

Chemical Synthesis

In this discovery, DAPA compounds were synthesized and tested on:

-   -   receptor activation assays    -   in vivo animal assays    -   human subjects with nasal discomfort.

From these studies four candidate API were identified that may haveutility in the relief of nasal discomfort from irritants, allergens, andinflammation.

The DIPA compounds were prepared by the following general method: 100 mL(23.7 g, ˜200 mmol) of isopropylmagnesium chloride (orsec-butylmagnesium chloride in the case of the di-sec-butyl derivatives)were obtained from Acros, as a 25% solution in tetrahydrofuran (THF) andplaced under nitrogen in a 500 mL flask (with a stir bar).Diethylphosphite solution in THF (from Aldrich, D99234; 8.25 g, 60.6mmol in 50 mL) was added drop-wise. After approximately 30 minutes, thereaction mixture warmed up to boiling. The reaction mixture was stirredfor an extra 30 min, followed by a drop-wise addition of the appropriaten-alkyl iodide solution in THF (from TCI; 60 mmol in 20 mL). Thereactive mixture was then stirred overnight at room temperature. Thereaction mixture was diluted with water, transferred to a separatoryfunnel, acidified with acetic acid (˜10 mL), and extracted twice withether. The ether layer was washed with water and evaporated (RotaVapBuchi, bath temperature 40° C.). The light brown oil was distilled underhigh vacuum [0.5 mm Hg]. The final products, verified by mass asdetermined by mass spectrometry, were transparent liquids that werecolorless or slightly pale yellow and had boiling points in the range of120 to 130° C.

Several samples of DIPA-1-7 or DIPA-1-8 were sent for detailed analysisby GC-MS (NCE Corporation, Pleasanton, Calif., USA,www.nceanalytical.com). Analysis was conducted on an Agilent GC/MSsystem 6890/5973 equipped with a TraceGold TG-624 column, with helium asthe carrier gas [flow rate: 1.6 mL/min] and the injector port set at220° C. [split ratio 50:1, temperature program: 100 to 240° C.]. The TIC[total ion chromatogram] showed the main components as having aretention time of 18 to 19 min, with the detected peaks accounting for97.2% of the total area. Similar results of 97 to 99% purity wereobtained with other samples. When gas chromatography [equipped with aflame ionization detector (Dong Wha Corporation, Seoul, Korea)] was usedas the analytical system, synthesized compounds were also found to be 97to 99% chromatographically pure.

The following compounds were prepared by this method wherein Table 1Aand Table 1B compounds are embodiments of the invention.

TABLE 1A Chemical structures of diisopropyl-analogs. Code Chemical NameChemical Structure DIPA-1-5 1-[Diisopropyl-phosphinoyl]- pentane

DIPA-1-6 1-[Diisopropyl-phosphinoyl]- hexane

DIPA-1-7 1-[Diisopropyl-phosphinoyl]- heptane

DIPA-1-8 1-[Diisopropyl-phosphinoyl]- octane

DIPA-1-9 1-[Diisopropyl-phosphinoyl]- nonane

TABLE 1B Chemical structures of di-sec-butyl-analogs. Code Chemical NameChemical Structure 2-4 1-[Di-sec-butyl-phosphinoyl]- butane

2-5 1-[Di-sec-butyl-phosphinoyl]- pentane

2-6 1-[Di-sec-butyl-phosphinoyl]- hexane

2-7 1-[Di-sec-butyl-phosphinoyl]- heptane

2-8 1-[Di-sec-butyl-phosphinoyl]- octane

3-1 1-[Diisobutyl-phosphinoyl]- pentane

3-2 1-[Di-sec-butyl-phosphinoyl]- 3-methyl-butane

General Observations on DAPA Compounds

DAPA compounds are colorless liquids with a density less than water [0.7to 0.8 g/cc]. They are generally soluble in water or saline at up to 20mg/mL, or for the compounds with greater than or equal to 16 carbonatoms, a homogeneous emulsion of very fine droplets. When DAPA compoundsare applied to the facial skin as an aqueous solution at 5 to 10 mg/mLthere is no irritation or blanching. For certain analogs, contacting thefacial skin with a solution at a concentration of 5-20 mg/mL produce asensation of strong cooling within 1 min especially when applied toperiorbital skin. The effects are strong on non-keratinzing tissues suchas the lining of the upper digestive tract and the ocular margins. Theeffects of these compounds after intranasal instillation have not beenreported in the literature.

The pharmacological effects of DIPA-1-8 and DIPA-1-9 are clearlydifferentiated from the sympathomimetic decongestants. DIPA-1-9 does notcause blanching of the skin or reduce redness from the blood vessels ofthe eyelids which are classical signs of α-adrenergic sympathomimeticactivity. These compounds have a mild cooling action on human nasalmucosa, an effect that is not seen with sympathomimetic decongestants.Sympathomimetic agonists such as clonidine do not interact do notactivate TRPM8 [Bavencoffe, A. et al. The transient receptor potentialchannel TRPM8 is inhibited via the α2A adrenoreceptor signaling pathway.J. Biol. Chem. 285, 9410-9419 (2010)]. The long durations of action ofDIPA-1-8 and DIPA-1-9 are also not seen with the standardsympathomimetic decongestants.

Note that the diisopropyl groups of the DIPA compounds of this inventiondo not have a chiral center but each of the sec-butyl groups incompounds of the Di-sec-butyl-phosphinoyl series has a chiral centre,and that each chiral centre may independently be in the (R) or (S)configuration. As a consequence, a compound such as 2-6 has fourpossible stereoisomers: two optically active stereoisomers (i.e., R,Rand S,S), and two optically inactive meso forms (i.e., R,S and S,R).Unless otherwise indicated, a reference to Di-sec-butyl-phosphinoylcompounds is intended to be reference to any one of the fourstereoisomers, and any mixture of any two or more of the fourstereoisomers. The absence of stereoisomers in the DIPA compounds is anadvantage in drug development over molecules containing sec-butyl groupsbecause current regulations often require that each enantiomer be eithersynthesized or isolated separately and then individually evaluated fortoxicological activities.

The effects of diisopropyl versus the di-sec-butyl congeners werestrikingly different in laboratory rats when given by the perioral ortopical routes. [Table 3]. Perioral or topical application of DIPAanalogs [DIPA-1-5, DIPA-1-6, DIPA-1-7] elicits vigorous shaking in thewhole animal, but this effect is hardly seen with the di-sec-butylcongeners. This is because DIPA-1-5, DIPA-1-6, and DIPA-1-7 are able topenetrate the membrane barriers in the gut and keratinized skin. Whengiven intravenously, the di-sec-butyl analogs are active.

Additional Terminology Used

Compositions: One aspect of the present discovery pertains to acomposition (e.g., a pharmaceutical composition) comprising a DAPAcompound, as described herein, and a pharmaceutically acceptablecarrier, diluent, or excipient. Another aspect of the present discoverypertains to a method of preparing a composition (e.g., a pharmaceuticalcomposition) comprising mixing a DAPA compound, as described herein, anda pharmaceutically acceptable aqueous solution. In one embodiment, thecomposition comprises the DAPA compound as an aqueous solution at aconcentration of 0.5-20 mg/mL. The composition may be provided withsuitable packaging and/or in a suitable container. For example, thecomposition may be provided as a manually activated sprayer or as nosedrops carrying a DAPA compound or a composition comprising a DAPAcompound.

Discomfort of the Nasal Cavity: In one embodiment (e.g., of use inmethods of therapy, of use in the manufacture of medicaments, of methodsof treatment), the treatment is treatment of sensory discomfort. In asecond aspect of treatment, the treatment is to provide immediatesymptomatic relief of nasal congestion, and thus to increase patientawareness and adherence to use of the medication. In the second aspect,in which the combination embodiment has a faster onset of action, thetreatment has prophylactic and therapeutic actions.

The term “sensory discomfort”, as used herein, relates to irritation,itch, pain, or other dysesthesias (abnormal sensations; such as“stuffiness”, congestion, obstruction, burning sensations, or feelingthe presence of a foreign body, or pins and needles) from the nasalcavity surfaces. The term implies activation of nociceptors located onsensory nerve endings of the body. Nociceptors are stimulated, forexample, by high or low temperatures, mechanical pressure, chemicals(e.g., capsaicin, acidity, pollutants, etc.), injury, inflammation, andinflammatory mediators. A compound, such as DIPA-1-8 or DIPA-1-9, thatdecreases sensory discomfort, can be termed an anti-nociceptive agent.

In one embodiment, the treatment is treatment of rhinitis and sinusitis.In one embodiment, the treatment is treatment of the symptoms of the“empty nose syndrome”. In one embodiment, the treatment is treatment ofnasal irritation from air pollutants. In one embodiment, the treatmentis treatment of heat discomfort. In one embodiment, the treatment istreatment of rhinosinusitis. In one embodiment, the treatment is torefresh breathing sensations, to reduce snoring, and to reduce sleepapnea. In one embodiment, the treatment is treatment of allergicrhinitis. In one embodiment, the treatment is treatment is to convey asense of refreshment to breathing in a human which can enhancemeditative exercises such as yoga.

Treatment: The term “treatment,” as used herein in the context oftreating a disorder, pertains generally to treatment of a human or ananimal (e.g., in veterinary applications), in which some desiredtherapeutic effect is achieved, for example, the inhibition of theprogress of the disorder, and includes a reduction in the rate ofprogress, a halt in the rate of progress, alleviation of symptoms of thedisorder, amelioration of the disorder, and cure of the disorder.Treatment as a prophylactic measure (i.e., prophylaxis) is alsoincluded. For example, use with patients who have not yet developed thedisorder, but who are at risk of developing the disorder, is encompassedby the term “treatment.” Treatment to enhance the basal levels ofcognitive awareness of the medication for the purposes of increasingadherence to the use of the medication is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Routes of Administration: The term “topical application”, as usedherein, refers to delivery onto surfaces of the nasal cavity in contactwith air.

Subject/Patient may be a mammal, for example, a canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig),ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., amonkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g.,gorilla, chimpanzee, orang-utang, gibbon), or a human. In one preferredembodiment, the subject/patient is a human.

Dosage: The dose and dosing regimen can be on an “as needed basis” oronce or twice daily, depending on the severity of the condition beingtreated. Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

Delivery Systems and Tissue Targets

The targets for drug delivery are the non-keratinizng stratifiedepithelium, such as the respiratory epithelium, covering the nasalcavity and the paranasal sinuses. Treatment is focused on theinflammatory processes underlying the rhinitis as the best way to treatdisorders of the nasal cavity.

The delivery of the DAPA compounds can be achieved with the compounddissolved in purified water or isotonic saline or phosphate bufferedsaline. For a liquid vehicle, a preferred concentration of the DAPAcompound is in the range of 0.5 to 10 mg/mL. A preferred amount of theDAPA compound delivered at the site of application is 0.5 to 5 mg.

Little is directly known about the neurophysiology of tissue targets inthe nasal cavity [except for olfaction]. The surface is innervated by abranch of the trigeminal nerve. The terminology for describing thesensations of the nasal surfaces is not standardized. For example,menthol lozenges cool the nasopharynx, but it is not clear if it coolsthe turbinates. Inhaled menthol vapors are pungent, but it is not clearif these sensations can be described as cool or cold. Breathing frigidair causes sting and rhinorrhea. Only breathing cool air, for example,at the seaside convey the right degree of coolness and the sense ofpatency. This sensation is pleasurable, but the exact air temperatureand humidity for capturing this sensation have not been defined.

There is a general view that “TRP-” ion channel receptors (A1, M8, andV1 to 4) are the principal physiological elements for physiologicaltemperature detection. The TRPM8 receptor is the one that responds tosensory/cooling agents such as menthol and icilin [McKemy et al.Identification of a cold receptor reveals a general role for TRPchannels in thermosensation, Nature, 416, 52-58, 2002]. TRPM8 is aprotein with 1104-amino acid residues and has six transmembrane domains.Activation of this receptor by lowering ambient temperature results inopening of pores of transmembrane loop and non-specific cation entryinto the cell. Depolarization of TRPM8 receptors on sensory neurons maythen transmit signals to the central nervous system primarily via AO(and some C) fibres.

While this concept for the role of TRPM8 in sensory physiology may bevalid for detecting physical changes in temperature, the interpretationof the sensory effects of chemical agents such as menthol and icilin aremore complex. Menthol not only stimulates TRPM8 in vitro, but alsoTRPV3, a receptor associated with warmth and glycinergic transmission[Macpherson et al. More than cool: promiscuous relationships of mentholand other sensory compounds. Mol Cell Neurosci 32:335-343, 2006:Sherkheli et al., Supercooling agent icilin blocks a warmth-sensing ionchannel TRPV3, Scientific World Journal, 2012: 982725, 2012:Cho et al.TRPA1-like channels enhance glycinergic transmission in medullary dorsalhorn neurons. J Neurochem 122:691-701, 2012]. Thus, menthol and icilinare called “promiscuous” agents and are not selective for one receptorprotein.

The Applicant has screened a large database of cooling agents forsensory effects on the skin [keratinizing] and the ocular rim[non-keratinizing] and found that there are distinct responsive elementsin the two types of epithelia [Wei. Sensory/cooling agents for skindiscomfort. Journal Skin Barrier Research 14: 5-12, 2012].

The epithelia of the nares and the anterior nasal vestibule arekeratinizing, but then transitions to respiratory epithelium[non-keratinizing]. When one examines the structure-activityrelationships (SAR) of the DAPA compounds [Formula 1] on keratinizedskin, it is noted that when R₁=R₂=isopropyl and R₃=n-hexyl (C₆) orn-heptyl (C₇), then strong penetrating cooling is observed. Cooling oflong duration is obtained on non-keratinizing epithelia with R₃=n-octyl(CO and n-nonyl (C₆). However, when R₁=R₂=sec-butyl dynamic cooling isobserved when R₃=n-pentyl to n-heptyl (C₅ to C₇). Thus, there are subtledifferences among the compounds embraced by Formula 1.

Shaking behavior is a rapid alternating contraction of the supinationand pronation muscles about the spinal axis, and is readily observed andcounted. Fur-coated and feathered animals—when wet and cold—shake, likea wet dog [Dickerson et al., Wet mammals shake at tuned frequencies todry. J. Royal Society, Interface 9, 3208-3218, 2012; Ortega-Jimenez, V.M. et al. Aerial shaking performance of wet Anna's hummingbirds. J.Royal Society, Interface 9, 1093-9, 2012; Wei, Pharmacological aspectsof shaking behavior produced by TRH, AG-3-5, and morphine withdrawal,Federation Proc. 40: 1491-1496, 1981].

“Wet-dog shaking” has been studied in detail in animals. Rats can shaketheir head, the upper torso, or the shaking can be sufficiently violentto affect the whole body and make the animal lose its balance. Thepurpose or survival value of shaking to fur-coated and featheredorganisms is to remove water droplets trapped on or near the skin.Removal of the water droplets on or near the skin by shaking reduces theorganism's need to expend energy to remove the water by evaporation. Thelikely equivalent behaviour to shaking in humans is shivering, acondition caused by generalized sensations of coolness/cold. Humansubjects recovering from the deep hypothermia of anaesthesia manifestvigorous shaking; a condition called post-anaesthetic shivering.

For the DAPA compounds the shaking frequency seen after intravenousinjection is a good approximation of the compound's potency forproducing cold when administered into the nasal cavity surfaces.

Test compounds were also evaluated on receptor assays: on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPRTETRA™) instrument. The specificity of the test compounds wereexamined on TRPV1 channels (human TRPV1 gene expressed in HEK293 cells)and TRPA1 channels (human TRPA1 gene expressed in CHO cells). ChanTestCorporation, 14656 Neo Parkway, Cleveland, Ohio 44128, USA, was thecontractor for these tests.

Selection of Active Ingredient

Ideally, an active pharmaceutical ingredient (API) formulated fordelivery to the nasal epithelium should be chemically stable, non-toxic,and sufficiently long-acting and potent to activate the mechanisms thatresult in a sense of refreshed breathing and a reduction of nasaldiscomfort. The API should be dissolved and evenly dispersed in acomposition so that during manufacture the formulation maintains aconstant concentration. The final product should meet standards ofcleanliness and sterility. For purposes of formulation, the API can be aliquid at standard conditions of temperature and pressure (STP) and thatis evenly dissolved in aqueous solutions at neutral pH and/orisotonicity. Sterility of the final product can be optimally achieved byusing purified reagents and filtration through micropore filters,heating, or irradiation. Standard excipients, such as preservatives, maybe added to optimize the formulations, but the important ingredientsshould be preferably soluble in aqueous media such as purified water,isotonic saline, or phosphate buffers.

For a given individual, the perceived sensation is a function of theparticular cooling agent, the dose, the vehicle used to carry thecooling agent, the method of topical delivery, and the nature of thetarget surfaces. The Applicant has screened a number of candidatecompounds, including diverse compounds such as p-menthane carboxamidesand icilin [Wei, 2012 vide supra]. The studies here identify DIPA-1-8,DIPA-1-9, 2-6 and 2-7 as having the preferred desired properties of anideal API for the nasal cavity epithelium to refresh, to reducerhinorrhea, to be compatible with formulations of an intranasal steroidor intranasal antihistamine, and to enhance patient adherence to use ofa topical intranasal medication. These analogs are selected because theyproduce mild cooling on nasal mucosa and do not produce excessivecooling on nasal skin.

A key factor to the successful management of nasal discomfort is thewater-solubility of the active ingredient for delivery to the nasalmucosa. A water-soluble API has tremendous advantages for ease and forhomogeneity of delivery to the target. If a chemical is not soluble inwater, solvents and excipients must be used. Then the solvent or matrixused has to be free of unpleasant effects on the nasal mucosa.

The applicant has screened a number of water soluble and water insolublecompounds and the results are described in Case Study 5. For watersoluble compounds such as CPS-030 [WS-30] [U.S. Ser. No. 13/261,061],(L)-Monomenthane-3yl carbonate [RightCool™ monomenthyl glutarate], and3-(1-Methoxy)propane-1,2-diol [Cooler 10] were tested. The goal was todetermine if such compounds can accelerate the sense of patency. It wasfound that CPS-030 at 5 mg/mL produced immediate sensations of coolness,but this was also felt on the skin of the nostrils, and there was asense of wetness. Monomenthyl glutarate and Cooler 10, which are watersoluble, were not active at 8 mg/mL Thus, CPS-030 could potentially beused as an enhancer of the DAPA compounds, to inform the patient that adrug had been delivered to the nose.

Water insoluble compounds may be useful for chronic rhinosinusitis orthe “empty nose syndrome” because these disease conditions arelong-lasting. With the correct formulations, e.g. milling and suspensioninto very fine particles or the incorporation of these compounds intogels, these water insoluble compounds, based on their chemical structureand reported pharmacological properties, are reasonable choices forprolonging duration of action if combined with DAPA compounds. These aresome candidate compound: icilin; CPS-125[2-isopropyl-5-methyl-cyclohexanecarboxylic acid[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide]; CPS-195[,2-Isopropyl-5-methyl-cyclohexanecarboxylic acid[2′-hydroxy-2′-(3″-hydroxy-phenyl)-ethyl]-N-methyl-amide]; Ax-8[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexane-carbonyl)-amino]-aceticacid isopropyl ester; Ax-10(R)-2-[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-propionicacid isopropyl ester;(1R,2S,5R)-2-isopropyl-5-methyl-N-(2-(pyridin-2-yl)-ethyl-cyclohexanecarbox-amide;(1R,2S,5R)-N-(4-(cyanomethyl)-phenyl)-2-isopropyl-5-methylcyclohexylcarboxamide, and “M8-Ag”(4-[5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-3-yl]morpholine [Patel,R. Anti-hyperalgesic effects of a novel TRPM8 agonist in neuropathicrats: A comparison with topical menthol. Pain 155, 2097-107 (2014)].

To summarize the design concepts that lead to the selection of the API,the delivery system, and the site of delivery, as being suitable for thepractice of the discovery:

-   -   DAPA compounds were identified that were soluble in water at up        to 20 mg/mL. These compounds are stable to heat, and exerted a        potent therapeutic effect on nasal discomfort and inflammation        at applied concentration of 1 to 10 mg/mL. In animal studies,        tachyphylaxis does not develop to repeat applications.    -   The receptor target for DAPA compounds was ascertained in in        vitro studies. The lead candidate was selective for TRPM8 and        not for TRPV1 or TRPA1.    -   The biological activity of DAPA compounds was defined in an        animal model of “wet-dog shakes”. The perioral, topical/dermal,        and intravenous activates were compared, and the selective        differentiation of diisopropyl analogs from di-sec-butyl analogs        was established.    -   In volunteers with rhinitis and nasal discomfort, the efficacy        of four compounds for reducing nasal discomfort and rhinitis was        established.    -   Compounds that produced strong cold on keratinized nasal skin,        e.g. DIPA1-7, were considered less desirable than compounds that        mildly refreshed the nasal mucosa [a non-keratinizing surface].        DIPA1-8 and DIPA1-9, were chosen as lead candidates.    -   Tests in human volunteers showed that four compounds, especially        DIPA-1-8 and DIPA-1-9, were effective for relieving sensory        discomfort from rhinitis and nasal pollutants. In normal        subjects, there was enhanced refreshed breathing.    -   Tests of combinations of the DAPA compounds of the preferred        embodiments showed that these compounds were compatible with        some standard formulations of intranasal steroids and intranasal        antihistamines.    -   Administration of these combinations showed that the combination        was the preferred nasal medication and increased patient        adherence to a dosage regimen.

Study 2 Results of TRPM8, TRPA1, and TRPV1 Receptor Assays:

The in vitro effects of test compounds were evaluated on cloned hTRPM8channel (encoded by the human TRPM8 gene, expressed in CHO cells) usinga Fluo-8 calcium kit and a Fluorescence Imaging Plate Reader(FLIPRTETRA™) instrument. To examine the specificity of the testcompounds, further tests were conducted on TRPV1 channels (human TRPV1gene expressed in HEK293 cells) and TRPA1 channels (human TRPA1 geneexpressed in CHO cells). The assays were conducted by ChanTestCorporation, 14656 Neo Parkway, Cleveland, Ohio 44128, USA.

Test compounds and positive control solutions were prepared by dilutingstock solutions in a HEPES-buffered physiological saline (HBPS)solution. The test compound and control formulations were loaded inpolypropylene or glass-lined 384-well plates, and placed into the FLIPRinstrument (Molecular Devices Corporation, Union City, Calif., USA). Thetest compounds were evaluated at 4 or 8 concentrations with n=4replicates per determination. The positive control reference compoundwas L-menthol, a known TRPM8 agonist. The test cells were ChineseHamster Ovary (CHO) cells stably transfected with human TRPM8 cDNAs.

For FLIPRTETRA™ assay, cells were plated in 384-well black wall, flatclear-bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine MultiwellCell Culture Plate) at approximately 30,000 cells per well. Cells wereincubated at 37° C. overnight to reach a near confluent monolayerappropriate for use in a fluorescence assay. The test procedure was toremove the growth media and to add 40 μL of HBPS containing Fluo-8 for30 minutes at 37° C. 10 μL of test compound, vehicle, or controlsolutions in HBPS were added to each well and read for 4 minutes.

Concentration-response data were analyzed via the FLIPR Control softwarethat is supplied with the FLIPR System (MDS-AT) and fitted to a Hillequation of the following form:

${RESPONSE} = {{Base} + \frac{{Max} - {Base}}{1 + \left( \frac{xhalf}{x} \right)^{rate}}}$

where: “Base” is the response at low concentrations of test compound;“Max” is the maximum response at high concentrations; “xhalf” is theEC₅₀, the concentration of test compound producing half-maximalactivation; and “rate” is the Hill coefficient. Nonlinear least squaresfits were made assuming a simple one-to-one binding model. The 95%Confidence Interval was obtained using the Graph Pad Prism 6 software.

Of the 12 compounds tested, all showed full efficacy on the TRPM8receptor, i.e., at higher tested concentrations there was ˜100%stimulation of calcium entry, and the data fitted a sigmoidaldose-response curve. The results for 10 of the compounds of thisinvention are illustrated in FIG. 1.

FIG. 1 is a graph of TRPM8 potency of Diisopropyl-phosphinoyl andDi-sec-butyl phosphinoyl alkane analogs in the in vitro TRPM8 assay.Units are in comparison to I-menthol potency in the same assay. Thenumbers on the abscissa represent the number of carbons in one of then-alkyl side-chain: namely, the 4-5-6-7-8-9 represents the butyl,pentyl, hexyl, heptyl, octyl, and nonyl group, respectively.

The EC₅₀ of the more potent compounds (DIPA-1-7, DIPA-1-8, DIPA-1-9,2-5, 2-6, 2-7, 2-8) fell within a narrow range with overlapping 95%Confidence Intervals. [Table 2] The potency of DIPA-1-7 and DIPA-1-8 aresimilar and significantly greater than the potencies of DIPA-1-5 andDIPA-1-6. By contrast the structural modifications of comparativecompounds 3-1 and 3-2 resulted in a significant loss of bioactivity.DIPA-1-10 was synthesized at a later time and tested by David Anderssonof King's College, London, U.K. It was found to be 2.4× less active thanDIPA-1-9 [or 1.7× menthol on Table 2]. The data for DIPA-1-10 is notincluded in the Table 2 because it was obtained under different assayconditions.

To examine the specificity of the test compounds, further studies wereconducted on TRPV1 channels (human TRPV1 gene expressed in HEK293 cells)and TRPA1 channels (human TRPA1 gene expressed in CHO cells). The testcells were Chinese Hamster Ovary (CHO) cells or Human Embyronic Kidney(HEK) 293 cells transfected with human TRPV1 or TRPA1 cDNAs. Thepositive control reference compound was capsaicin (a known TRPV1agonist) or mustard oil (a known TRPA1 agonist). DIPA-1-7 and DIPA-1-8did not exhibit any agonist on antagonist activity on TRPA1 channels atmaximum tested concentrations of 100 μM. The results for DIPA-1-8 areshown in FIG. 2.

FIG. 2 shows the lack of agonist activity of DIPA-1-8 in cellstransfected with TRPV1 or TRPA1 plasmids. The positive controlscapsaicin and mustard oil for TRPV1 and TRPA1 are active, but DIPA-1-8is not. The ordinate is given in Relative Fluorescence Units; % ofmaximum, which measure calcium entry into the transfected cells and theabscissa is the logarithm of the concentration of the test compound.DIPA-1-8 was also devoid of antagonist activity against TRPV1 or TRPA1.

The EC₅₀ values do not give information on the quality of the heatabstraction sensation in the nasal cavity, on the duration of action, oron the accessibility [distribution] of the molecule to tissue targetssuch as the nasal mucosa. The EC₅₀, however, gives guidance on therelative potencies of the different analogs. Of special importance isthe differentiation of the drug effect on the keratinized epithelia ofthe nose [nostril skin and vestibule] versus the non-keratinizingepithelia of the nasal mucosa. The use of the aqueous solution on theswab makes the drug available to all epithelia. The identification ofagents that are optimized for the nasal mucosa requires bioassays thatdirectly address these questions. Part of the discovery process here isthe recognition that stimulation of cold receptors on the keratinizedsurfaces is not desirable, whereas stimulation of the mucosal receptorsgives the desired drug effect. Both responses are likely to be mediatedby the TRPM8 receptor protein and correlated to the EC₅₀. The presenceof 15 to 16 carbons in molecules of Formula 1 appear to optimize thelocalization and distribution of the drug candidate to the nasal mucosa.

TABLE 2 EC₅₀ and relative potency of compounds on TRPM8.. 95% ConfidenceRelative Code EC₅₀ μM Interval Potency Menthol 3.8 2.5 to 5.6 1.0DIPA-1-5 5.6 4.4 to 7.2 0.7 DIPA-1-6 2.4 1.5 to 4.0 1.6 DIPA-1-7 0.7 0.5to 1.0 5.4 DIPA-1-8 0.7 0.5 to 1.0 5.4 DIPA-1-9 0.9 0.4 to 2.5 4.0 2-414.5  7 to 29 0.3 2-5 1.7 1.0 to 2.9 2.2 2-6 0.8 0.5 to 1.3 4.7 2-7 1.10.6 to 2.3 3.4 2-8 1.3 0.7 to 2.3 2.9 3-1 24  8 to 76 0.2 3-2 4.2  1.6to 10.8 0.9

Study 3 Activity in Laboratory Rat: Perioral, Topical and IntravenousDelivery

To get a better idea on the in vivo activity of these DAPA compoundsfurther studies were conducted on the laboratory rat afteradministration of the test compounds by three different routes:intravenous, perioral, and topical. Variation on the routes ofadministration provides information on the ability of the molecule tocross membrane barriers.

Fur-coated and feathered animals—when wet and cold—shake, like a wet dog(see, e.g., Dickerson et al., 2012; Ortega-Jimenez et al., 2012; Wei,1981). These shakes are rapid alternating contractions of the supinationand pronation muscles about the spinal axis, and can be readily observedand counted. “Wet-dog shaking” has been studied in detail in animals andthis behavior is interpreted to have survival value because shaking, byremoving the water off t skin, reduces the need to expend evaporativeenergy to remove wetness. The triggering sensation for shaking is thushaving water trapped in between hair follicles or feathers. Humans havelittle hair on skin and do not shake. The likely equivalent behavior toshaking in humans is shivering, a condition caused by generalizedsensations of coolness/cold and wetness.

Drug-induced shaking in animals has been reviewed (see, e.g., Wei,1981). Under the right conditions, drug-induced shaking can be observedin the pentobarbital-anesthetized rat, enhanced by hypothermia and cold,and inhibited by elevating body temperature.

In experiments conducted here, test compounds were evaluated for“wet-dog shaking” as a model of cooling sensation. Using a standardizedprocedure, test compounds were compared in their ability to stimulatethe shaking response by perioral administration, by topical delivery tothe abdominal skin, and by intravenous administration through acannulated femoral vein.

Perioral. Test compounds were dissolved in saline and administered byoral gavage to pentobarbital-anesthetized male albino rats at 20 mg/kgat a volume of 0.1 mL/100 g body weight [n=3 to 4 rats per compound].Shaking was counted over a 40 min period and recorded at 10-minintervals. The results are shown in Table 3.

Three of the four “diisopropyl” compounds caused vigorous shaking. The“di-sec-butyl” compounds were relatively inactive, except 2-5 whichelicited an average of 4 shakes in the 40 min observation period. Bycontrast, DIPA-1-5, DIPA-1-6, and DIPA-1-7 produced an average shakingfrequency of 86, 56, and 36 shakes, respectively. The strong activity ofDIPA-1-5 was unusual. Applied to the skin, DIPA-1-5 has a refreshingcooling sensation, but the duration of action of only about 30 min wassignificantly less than that for DIPA-1-6 and DIPA-1-7. It is possiblethat its smaller molecular size facilitates absorption and allowsgreater access to systemic receptors, and therefore more shaking.

The relationship of the shake response to temperature sensation wasfurther studied [in pentobarbital-anesthetized rats]. After injection ofthe sodium pentobarbital anesthetic, rectal temperature drops, andreaches approximately 35° C. in about 10 min. This hypothermia can bereversed by placing the animal on a heated surface and body temperaturemaintained at 38° C. DIPA-1-7 20 mg/kg perioral elicited 36±5 shakes(n=6) in the anesthetized rat, but in the heated animals, the shakingfrequency was significantly reduced to 5±2 shakes (n=6) [P<0.001]. Thereduction of shaking frequency by ⅔ under heat indicated that the shakeresponse was linked to cold sensations and shivering.

Topical. Shaking is an excellent indicator of in vivo effect. Methodswere developed to determine if shaking was seen after topicalapplication of DAPA compounds. The abdominal skin of thepentobarbital-anesthetized rat was shaved and 20 μL of the pureunadulterated DIPA chemical was applied with a micropipette on a ˜1 cmdiameter circle of skin, enclosed with a ring of cream [Baby cream“Nevskaya kosmetika Detskyi” Nevskaya Kosmetika Inc., Saint-Petersburg192029], The number of shakes was counted for 1 hr after application.

The results for topical, perioral, and intravenous responses aresummarized in Table 3. The surprising potency of DIPA-1-5 and DIPA-1-6was unexpected but similar to what was seen with perioraladministration. These smaller molecules may penetrate faster through theskin barrier and go into the systemic circulation. However, the value ofthis fast action is uncertain. In most contemplated topical applicationsof this discovery, the preference is for the drug action to remainlocalized and not systemic.

Intravenous. When the relative activities of the analogs for producingshaking by the perioral and topical routes were compared to the EC₅₀ forTRPM8 activation [as inversely measured by the xMenthol potency] it canbe seen that the two variables are not correlated. For example, 2-6 is4.7× menthol, but does not produce shaking by perioral or topicaladministration. Yet DIPA1-7, which 5.7× menthol, produces vigorousshaking by these routes. The lack of quantitative correlation isperplexing, because it would be expected that the cooling properties arelinked to TRPM8 activation. To clarify the discrepancy, the testcompounds were compared by the intravenous [i.v.] route ofadministration, a delivery route which is less influenced by membranebarriers.

Male rats weighing ˜220-240 g were anesthetized with sodiumpentobarbital, 55 mg/kg intraperitoneal, and after the loss of therighting reflex, animals were placed on a heated table and bodytemperature maintained at 37 to 38° C. The femoral vein was cannulatedwith PE-20 tubing connected to a 1 mL syringe. Stock solutions wereprepared in normal saline at 10 mg/mL and further diluted to 2 mg/mL onthe day of the experiment and injected at 0.1 mL/100g body weight togive a dose of 1 mg/kg i.v. There were n=3 to 6 per test substance.Shaking frequency was counted for 30 min after i.v. delivery and theresults compared with the Student's t-test. Two trials were conductedper animal with a 10 to 15 min interval between doses. The shakingfrequency after intravenous injections is shown in Table 3.

Shaking was observed immediately after i.v. injection and at least 78%of the total shakes occurred in the first 5 min after injection. Theresponse in the second trial was at least as robust in the first trial,showing the lack of desensitization [FIG. 3]. The greater response inthe second trial may be due to cumulative effects or a lightening ofanesthesia.

FIG. 3. shows the absence of tachyphylaxis [desensitization] to theshaking effects of repeated injections of DIPA-1-7, 2 mg/kg intravenous,at 15 min intervals. It can be seen that the shaking intensity of the3^(rd) and 4^(th) trials were not diminished by the previous injections.

TABLE 3 Shaking frequency after perioral [per 20 mg/kg body weight] ortopical delivery of 20 μl test compounds [per animal] or intravenously[2 mg/kg] to the anesthetized rat. Mol Code Wt # Cs x Menthol PerioralTopical Intravenous DIPA-1-5 204 11 0.7 86 ± 7 138 ± 15 19 ± 3 DIPA-1-6218 12 1.6 56 ± 5 69 ± 8 39 ± 4 DIPA-1-7 232 13 5.4 36 ± 4 79 ± 8 25 ± 3DIPA-1-8 246 14 5.4 0  7 ± 2 14 ± 2 DIPA-1-9 260 15 4.0 0 0  3 ± 1 2-4218 12 0.3 0 0  8 ± 2 2-5 232 13 2.2  4 ± 1 0 20 ± 2 2-6 246 14 4.7 0 030 ± 3 2-7 260 15 3.4 0 0 15 ± 2 2-8 274 16 2.9 0 0 2.

Interpretation of EC₅₀ and Shaking Data After Perioral, Topical, andIntravenous Delivery

The strong bioactivity of intravenous 2-6 and 2-7 is in sharp contrastto the results seen after perioral or topical delivery when no shakingwas observed. These results provide strong objective laboratory evidencethat the DIPA compounds of Table 1A are qualitatively different from thecorresponding di-sec-butyl compounds. The diisopropyl compounds with theshorter R₃ chain produce shaking by all three routes of administration,whereas the di-sec-butyl compound is active only by intravenousdelivery.

The TRPM8 EC₅₀ and perioral, topical, and intravenous provides anexcellent framework and rationale for the selection of the best API fornasal discomfort.

-   -   The less potent candidates on the TRPM8 EC₅₀ , namely, 1-5, 1-6,        2-4 and 1-10 were judged to be less suitable because of a lack        of activation power.    -   The perioral and topical shaking seen with 1-5, 1-6 and DIPA-1-7        made these candidates less attractive because shaking is        elicited by strong sensory stimuli, and this is not desirable in        the nasal cavity. The extra penetrating quality of DIPA-1-7 may,        however, be useful in congested situations such as the common        cold and severe sinusitis.    -   The lack of activity of DIPA-1-8, DIPA-1-9, 2-6 and 2-7 on        perioral and topical administration made these analogs        attractive because it meant that these molecules remained        localized in tissues after administration.    -   The greater shaking frequency see with 2-6 and 2-7 versus        DIPA-1-8 and DIPA-1-9 meant that these molecules produced        stronger sensations of cold, an effect which was confirmed on        nostril skin.

From this analysis of the four measurements, namely, TRPM8 EC₅₀ andperioral, topical, and intravenous shaking activity, the logical initialchoices for nasal discomfort is DIPA-1-8 and DIPA-1-9, followed by 2-6and 2-7. DIPA-1-9 is especially attractive because it does not causeshaking, and yet is potent on the TRPM8 receptor.

Study 4 Screening of Compounds and Case Studies in Human Subjects

In these studies, subjects were given dosages units containing 1.5 to1.75 mL of DAPA compounds stored in 2.0 mL microcentrifuge tubes (NovaBiostorage Plus, Canonsburg, Pa. 15317) and cotton swab (Puritan largecotton tipped applicators, Model 803-PCL) or SwabDose units, made byUnicep Corp., containing 2 mg/mL of DIPA-1-9. The tested compounds wereas a solution in distilled water or in 1 mL of purified USP water. Therange of tested concentrations was 1 to 4 mg/mL. The subjects were giveninstructions on how to place the tip of the applicator in the nostril,to gently compress the nostril with the thumb and forefinger todistribute the liquid into the anterior nasal vestibule. Approximately0.03 mL to 0.06 mL is delivered by this method of application to thenasal cavity. A larger volume was occasionally delivered if the subjectexcessively wetted the cotton tipped applicator.

In a second set of experiments, the delivery system used was nose drops.Solutions were made up 0.1 to 5 mg/mL in isotonic saline and placed in 4mL Nalgene 2752-9125 bottles which have a ½′ tip than can be insertedpast the nasal vibrissae, into the anterior nasal vestibule. With thehead tilted slightly backwards, squeezing the bottle will reliablydeliver ˜40 μL per drop into the pocket formed at the base of the nares.Two to three drops were delivered per nostril. For example, for a 2mg/mL DAPA compound the estimated delivered dose to both nostrils of thenose is 2 mg/mL×0.16 mL, or approximately 320 μg. This is comparable tothe potency of intranasal steroids used for allergic rhinitis: forexample, 55 μg of triamcinolone acetonide is delivered per actuation ofa spray bottle of Nasacort® and the recommended dosing procedure is twoactuations per nostril.

Three subjects, two with defined seasonal allergic rhinitis and onesubject with chronic rhinitis of unknown cause, volunteered to besubjects for testing for multiple sessions. The compounds in Table 1were tested when the subjects were symptomatic: i.e. were sneezing orhad rhinorrhea, or had itchy sensations in the nose and eyes. Thesubjects refrained from the use of any antihistamine or intranasalsteroids during the test periods, which lasted for six weeks. Subjectswere instructed to rinse the nose with water if there was any nasaldiscomfort: however, irritation and discomfort was not seen in thesetrials.

These observations were made. The DIPA compounds 1-8 and 1-9(1-[Diisopropyl-phosphinoyl]-octane and1-[Diisopropyl-phosphinoyl]-nonane]), respectively, produced immediate,robust, and penetrating feeling of nasal clearing. The passage of air inthe nostrils was refreshing and there was an absence of obstruction. Forsome subjects, sneezing and intranasal itching was inhibited within 5 to10 min after application, and these effects were long-lasting: for 12 hror more after a single instillation. But if the sneezing was on-goingbefore instillation, the sense of clean breathing may also potentiatethe sensation of itching in the nostrils. The consensus subjectivefeeling was, however, that nasal cavity discomfort was reduced andbreathing felt clear and normal. The strong, efficacious drug action isunusual and has not been previously recognized to be achievable, and wassurprising and amazing.

The two 1-[Di-sec-butyl-phosphinoyl]-alkanes of equal molecular weightand total number of carbons [14 and 15] to DIPA-1-8 and DIPA-1-9,namely, 2-6 and 2-7 1-[Di-sec-butyl-phosphinoyl]-hexane and1-[Di-sec-butyl-phosphinoyl]-heptane], respectively, also exhibitedefficacy. 2-6 and 2-7 had a stronger cooling action on the nasal mucosaand on the nostril skin after application. At higher doses, 2-6 and 2-7sometimes triggered sneezing and long-lasting cooling on the skin on thetip of the nose. These side-effects may limit their use. But if nasaldiscomfort is not controlled by DIPA-1-8 or DIPA-1-9, then 2-6 and 2-7are good back-up compounds.

For the other compounds, with total carbons numbers different from 14 or15, good control of the symptoms of rhinitis was not achieved. For thetwo analogs with 13 carbons, DIPA-1-7 and 2-5, robust sensations werefelt on the nostril skin and the nasal mucosa, but the beneficialeffects on rhinitis were not evident. Both molecules seemed to inducerhinorrhea in some trials, perhaps reflecting a strong sensory action oncold receptors or on serous glands. For the two analogs with 16 carbons,1-10 and 2-8, cooling sensations were minimally present afterinstillation, but there was little evidence of reducing the symptoms ofrhinitis. In one subject 2-8 appeared to increase “stuffiness” at thehigher concentration of 4 mg/mL.

These results of the various analogs, summarized in Table 4, areconsistent with the TRPM8 EC₅₀ data, and the shaking data seen afterintravenous, perioral and topical administration. The diminishedactivities of 1-10 and 2-8 are consistent with a lack of potency onTRPM8. DIPA-1-7 is very active on cold sensations and TRPM8, but itsability to penetrate and distribute in tissues is reflected in itsshaking activity after perioral and topical administration. This isundesirable because the molecule will move away from its intended targetsite. DIPA-1-7 and 2-5 are both smaller molecules, having 13 carbons,and have greater mobility in tissues. Thus, DIPA-1-7 and 2-5 act onkeratinized skin and do not localize well in the nasal cavity, and thislimits efficacy.

The four compounds with 14 or 15 carbons, namely DIPA-1-8, DIPA-1-9,2-6, and 2-7, are potent on TRPM8 and do not produce shaking afterperioral or topical administration. This means that the drug is potentand also remains localized at its site of application: a highlydesirable characteristic. Shaking may represent the ability of themolecule to produce a sensation of “stinging cold”, and the diminishedactivity of DIPA-1-9 on this end-point makes it the lead candidate.DIPA-1-8 is the second lead candidate, followed by 2-6 and 2-7. The moreintense cold seen with 2-6 suggests that it may be a preferred candidatefor modifications of conditions such as sinusitis, heat stress, and“night sweats”. In these conditions, a stronger sense of cold may havebetter therapeutic value.

In summary, the pattern of activity of each molecule is a sum ofpenetration, distribution, localization, and intrinsic activity at thereceptor. The best compounds are those that have potency on the nasalmucosa, and not on the keratinized skin of the nares and nasal vestibuleThe best compounds of the present discovery for nasal discomfort areDIPA-1-8 and DIPA-1-9, and 2-6 and 2-7, and are examples of1-[Dialkyl-phosphinoyl]-alkanes [(O═)PR₁R₂R₃] wherein each of R₁, R₂, iseither isopropyl or sec-butyl and R₃ is a linear alkyl group of 6 to 9carbons, and wherein the preferred embodiments have 15 or 16 carbons.

Case studies are described below which illustrate the use of severalDAPA compounds delivered to the nasal cavity. The preferred embodimentsare effective:

-   -   to reduce the discomfort of allergic rhinitis;    -   to reduce the discomfort of vasomotor rhinitis;    -   to reduce sneezing and rhinorrhea;    -   to enhance the sense of breathing fresh air in normal subjects;    -   to reduce the discomfort of breathing polluted air;    -   to enhance breathing comfort and performance in a professional        athlete;    -   to help an individual cope with heat stress; and    -   to reduce the severity of “night sweats” in a subject.

TABLE 4 Test Results of Compounds on Nasal Discomfort Caused by RhinitisChemical Code No. Carbons Mol.Wt. Efficacy Side-effects DIPA-1-7, 2-5 13232.34 + cold, rhinorrhea DIPA-1-8, 2-6 14 246.37 +++ cold skin for 2-6DIPA-1-9, 2-7 15 260.40 +++ cold skin for 2-7 DIPA-1-10, 2-8 16 274.44 0congestion

Study 5 Combination of DAPA Compounds and Medications in VariousIntranasal Sprays.

The primary goals of these experiments were to:

-   -   Determine if the selected cooling agent was chemically        compatible with the nasal medication containing an intranasal        steroid or intranasal antihistamine: that is, if the two items        could be homogeneously mixed in solution without the appearance        of precipitates. Other intranasal medications, such as isotonic        and hypertonic saline, cromolyn sodium and phenylephrine HCl,        were also examined.    -   Find a concentration and regimen of the selected cooling agent        in the combination that will impart a rapid strong refreshing        sensation capable of relieving the sense of nasal congestion.        This study was first conducted in individuals without rhinitis        and then in individuals with symptoms of allergic rhinitis.    -   Ascertain if the selected cooling agent in rhinitis patients        will improve adherence in the use of the intranasal steroid or        intranasal antihistamine and improve treatment outcome.

Standard approved over-the-counter intranasal steroids in the USA areNasacort®, Flonase®, and Rhinocort® containing the active ingredients oftriamcinolone acetonide, fluticasone propionate (or furoate), andbudenoside, respectively. These OTC bottles or their internationalequivalent were obtained and a cooling agent was added to thepreparation and the bottle coded. The bottles were then shaken andexamined to ensure thorough mixing of the contents. The test solutions,for example, of triamcinolone acetonide preparation had a cloudy/milkyappearance, thus mixing was examined under intense light to ascertainhomogeneous distribution of the cooling agent when added into solution.The concentrations of the cooling agent chosen for combination rangedfrom 1 to 10 mg/mL for the final concentrations selected as shown in theTables. The amphiphilic characteristic of the preferred embodiments gavethe mixed solution a bubbly appearance which was not seen with thevehicle control (water). These bubbles further confirmed the presence ofcooling ingredient in the combination.

Subjects without rhinitis given the coded preparations (with or withoutcooling agent) were asked to rate the medication on tastes, quality andintensity of cool sensations, and the feeling of nasal patency. Thesetests were then repeated in patients with allergic rhinitis. For theindividuals with rhinitis, a standardized total nasal symptom score(TNSS) questionnaire was administered every two days, and the bottlesweighed at that time. These procedures were repeated over a total 5-dayperiod of study. The weights of the bottle (weights of medicationsconsumed or WMC) measure the number of actuations that were used by thesubject and indicate adherence/compliance. The five DAPA compoundsselected for study were DIPA-1-7, DIPA-1-8, DIPA-1-9, DAPA-2-6, andDAPA-2-7. A standard questionnaire was used to record the TNSS at twoday intervals.

Combination of DAPA Compounds and Intranasal Steroids

The tested intranasal steroids were commercial formulations oftriamcinolone acetonide and of beclometasone dipropionate. The dosagerecommendations for these formulations are 2 actuations of the nasalpump once daily for triamcinolone acetonide, and 1 actuation ofbeclometasone dipropionate 2 times per day. The results are shown inTable 5 and 6. One can conclude that a DAPA cooling agent, added to astandard formulation of an intranasal steroid spray, imparts animmediate sense of refreshing coolness in the nasal cavity.

TABLE 5 Test of Intranasal Steroid Spray with Cooling Agent Adjunct*Agent Steroid Effects Vehicle** Triamcinolone No sensations of coolnessor Ac*** refreshment were reported after use of the spray. DIPA-1-9Triamcinolone Refreshing cool/cold were  5 mg/mL** Ac*** immediatelyobtained in the nasal cavity, lasting ~0.5 to 0.75 hr. No discomfort wasreported and no rhinorrhea was observed in the four tested subjects.DIPA-1-9 Triamcinolone Robust stinging cool/cold 10 mg/mL** Ac***sensations were obtained in the nasal cavity, lasting ~1 hr. There wasrhinorrhea in two of the four tested subjects. *Tests were conducted in4 normal volunteers without rhinitis. Subjects sprayed the preparationonce a day for 3 consecutive days and filled in a questionnaire formeach day with comments on the observed drug actions. **The vehicle wasdistilled water (0.1 mL). The test materials were added 50 mg or 100 mgdirectly to the liquid in the nasal spray bottle. ***Triamcinolone NasalSpray −24 hr, made by XingYin Company in NanJing China, registrationnumber 20020360. Each bottle contained 11 mg of triamcinolone acetonidein a 10 mL liquid composition. The total dose per two actuations perboth nostrils was 220 μg or equivalent to a total dose of 0.2 mL of thesolution, or 0.1 mL per nostril. Actual measurements of volume per twoactuations showed a range of 0.095 to 0.011 mL with an average of 0.099mL ± 0.001 (SD, n = 200 actuations).

TABLE 6 Test of Intranasal Steroid Spray with Cooling Agent Adjunct*Agent Steroid Effects Vehicle** Beclometasone No sensations of coolnessor dipropionate*** refreshment were reported after use of the spray.DIPA-1-8 Beclometasone Refreshing cool/cold sensations were 5 mg/mL**dipropionate*** immediately obtained in the nasal cavity, lasting ~0.5to 0.75 hr. No discomfort was reported and no rhinorrhea was observedthe four tested subjects. DIPA-1-9 Beclometasone Refreshing cool/coldsensations were 5 mg/mL** dipropionate*** immediately obtained in thenasal cavity, lasting ~0.5 to 0.75 hr. No discomfort was reported and norhinorrhea was observed the four tested subjects. *Tests were conductedin 4 normal volunteers without rhinitis. Subjects sprayed with oneactuation twice a day for 3 consecutive days and filled in aquestionnaire form each day with comments on the observed drug actions.**The vehicle was distilled water (0.1 mL). The test materials wereadded 50 mg directly to the milky/cloudy liquid in the nasal spraybottle. Rinoclenil ® contains phenylethylalcohol as an excipient.***Beclometasone dipropionate Rinoclenil ® Nasal Spray, made by Chiesi,Parma, Italy, registration number 3400937071024. One actuation pernostril, twice daily is expected to deliver 100 μg of beclomethasonedipropionate. A similar product, called Humex, was also tested withsimilar results.

Combinations of DAPA Compounds and Intranasal Antihistamine

Azelastine HCl is an effective intranasal antihistamine. The completemiscibility of the DAPA compounds in the colorless liquid of acommercial preparation of azelastine chlorohydrate (Allergodil®) wasnoted, and showed compatibility of the combination of the twoingredients. The strong bitter tastes of azelastine sprays have beenfound in previous studies of this topical antihistamine (Seidman et al.vide supra) but are not observed with the intranasal steroid sprays.Surprisingly, the cooling agents tested here, especially DIPA-1-9,reduced the intensity of the azelastine bitterness (Table 7). Thiseffect was also found when the spray was applied directly into the oralcavity. The acute bitterness was attenuated, although there was somelingering bitterness afterwards. The azelastine spray was well-acceptedin rhinitis patients and reduced the TNSS scores, especially in thecategory of nasal itching. From these results, one can conclude that aDAPA cooling agent can be added to a standard formulation of anintranasal antihistamine spray to impart an immediate sense ofrefreshing coolness in the nasal cavity.

TABLE 7 Test of Intranasal Antihistamine Spray with Cooling AgentAdjunct* Agent Antihistamine Effects Vehicle** Azelastine No sensationsof coolness or refreshment HCl*** were reported after use of the spray.In three out of four subjects, a strong bitter taste was noticed on thetongue. DAPA-2-6 Azelastine Refreshing cool/cold sensations were 5mg/mL** HCl*** immediately obtained in the nasal cavity, lasting ~0.5 to0.75 hr. No discomfort was reported and no rhinorrhea was observed thefour tested subjects. DIPA-1-9 Azelastine Refreshing cool/coldsensations were 8 mg/mL** HCl*** immediately obtained in the nasalcavity, lasting ~1 hr. No discomfort was reported and no rhinorrhea wasobserved the four tested subjects. Surprisingly, no subjects reportedbitter taste. *Tests were conducted in 4 normal volunteers withoutrhinitis. Subjects sprayed with one actuation twice a day for 3consecutive days and filled in a questionnaire form each day withcomments on the observed drug actions. **The vehicle was distilledwater. The test materials were added ~120 mg directly to the clearliquid in the nasal spray bottle (~13 mL) Allergodil ® is a clearcolorless liquid and completely miscible with DIPA-1-9 at the testedconcentration of 9 mg/mL of DIPA-1-9. ***Azelastine chlorohydrateAllergodil ® Nasal Spray, made by Meda Pharma, Paris, France.registration number 56FR22025007-00. One actuation per nostril isexpected to deliver 0.125 mg/dose of active substance. The singleactuation delivered ~0.135 mL of liquid.

Combination of DAPA Compounds With Intranasal Saline

Distilled water administered into the nasal cavity produces an acutesense of discomfort, most likely because of an osmotic effect on thesensitive cells of the nasal membranes. The unpleasant effect isimmediate and noxious, but does not last more than a few minutes. It issimilar to getting water into the nose in a swimming pool. Isotonicsaline or hypertonic saline, containing 0.9 g or 2.65 g of sodiumchloride per 100 mL of water, respectively, do not produce this osmoticeffect and are used as nasal sprays and rinses. The saline solutionssprayed into the nasal cavity do not irritate the nasal membranes orproduce cooling sensations. The efficacy of these saline solutions onthe treatment rhinitis is not robust, but have a flushing and cleansingaction of the nasal cavity.

Here a range of DAPA compounds 0.5 to 5 mg/mL were added to irrigationsolutions of isotonic or hypertonic saline and administered as a sprayin single or double actuations of 0.1 mL per actuation, or administeredas a nose drop using eye dropper bottles with an extended tip thatreliably releasing 0.04 mL per drop per squeezing. For the nose drops,the number squeezed into the nostril was 2 to 3 drops, or about 0.1 mLper nostril. The results are shown in Table 8.

The DAPA compounds administered via spray or drops in saline solutionsall produced immediate refreshing and clearing sensations in the nasalcavity. All subjects opined that breathing was immediately cleared,within 0.5 to 2 min of dosing. This was seen in normal subjects and insubjects with allergic rhinitis. The duration and intensity of coolnessvaried with dose and compound. DIPA1-7 had the fastest onset and coolingintensity, but at 5 mg/mL, it was also produced a mild degree ofrhinorrhea. DIPA-1-9 had the longest duration of action, for severalhours, and in some subjects using nose drops the sense of clearancelasted the whole day after administration. Case studies are furtherdetailed below. It is apparent, that addition of a DAPA compound to asaline composition, containing 0.9 to 2.65 g of sodium chloride per 100mL water mixes well and provides immediate relief for a congested nose.A robust cooling effect can be obtained by spray or by nose drops. TheDAPA compounds, at the tested concentrations of 0.5 to 5 mg/mL, arecompletely miscible and compatible with the saline solutions.

TABLE 8 Testing of Intranasal Irrigation Solutions with Cooling AgentAdjunct* Irrigation Agent solution Effects Vehicle** Distilled Distilledwater alone produced an immediate water*** sense of irritation anddiscomfort in four out of four subjects. Testing of this solution wasdiscontinued. DAPA-1-8 Isotonic Robust cooling was felt but nodiscomfort 5 mg/mL + saline*** was reported and no rhinorrhea was NaCl,0.9% observed in the four tested subjects. DAPA-1-9 Hypertonic Robustcooling was felt but no discomfort 5 mg/mL + saline*** was reported andno rhinorrhea was observed NaCl, 2.65% in the four tested subjects.*Tests were conducted in 4 normal volunteers without rhinitis. Subjectssprayed with two actuations twice a day for 3 consecutive days (for theisotonic and hypertonic saline experiments) and filled in aquestionnaire form each day with comments on the observed drug actions.**The vehicle was distilled water. Sodium chloride was added to thedistilled water to make up the isotonic or hypertonic saline. Ayr ®allergy and sinus hypertonic saline nasal mist is an example of aproduct line in the “saline” category and contains 2.65% sodiumchloride. The DAPA compounds are completely soluble in saline at thetested concentrations of 1 to 5 mg/mL. Saline nose drops containing DAPAcompounds could also be used with immediate onset of cooling. ***Oneactuation per nostril delivered volume of ~0.106 mL of saline solution.

Combination of DAPA Compounds With Other Intranasal Medications

Cromolyn sodium is a “mast cell stabilizer”, i.e. it inhibits therelease of inflammatory mediators from the mast cell, principallyhistamine. Histamine in the nasal cavity causes itch and sneezing andincreases blood flow and vascular permeability that lead to rhinorrhea.Cromolyn sodium is available as an OTC drug, with 2.5 mg delivered peractuation. The recommended procedure, e.g. for NasalCrom®, is to spray 3to 4 times day, with a maximum of 6 times. On the package insert, it isnoted that users of NasalCrom® may take several days to notice abeneficial effect and full effect is not seen until 1 to 2 weeks.Cromolyn sodium is considered active and safe, but it is not the drug offirst choice for allergic rhinitis because of its perceived limitedefficacy (Seidman et al. vide supra). Experimentally, it is shown herethat two DAPA compounds, added to NasalCrom® is chemically miscible, andthat an immediate cooling effect is obtained after spraying.NasalCrom®alone does not produce cooling. The results are shown in Table9.

TABLE 9 Testing of Intranasal Mast Cell Stabilizer with Cooling AgentAdjunct* Mast Cell Agent Stabilizer Effects Vehicle** NasalCrom ®*** Noirritation or discomfort was reported after use of the spray in four outof four subjects. There were also no sensations of coolness. DAPA-2-7NasalCrom ®*** No discomfort was reported and no 5 mg/mL rhinorrhea wasobserved in the four tested subjects. Immediate sensations of cooling orcold were noted. DAPA-2-6 NasalCrom ®*** No discomfort was reported andno 5 mg/mL rhinorrhea was observed in the four tested subjects.Immediate sensations of cooling or cold were noted. *Tests wereconducted in 4 normal volunteers without rhinitis. Subjects sprayed withone actuation three times a day for 3 consecutive days. **The vehiclewas distilled water. DAPA-2-5, DAPA-2-6 was added to NasalCrom ®, apreparation containing cromolyn sodium, benzoalkonium chloride, edetatedisodium, and purified water. The NasalCrom ® solution was a clear andtransparent liquid. The DAPA compounds were completely soluble in theNasalCrom ® at the tested concentrations of 2 to 5 mg/mL. ***Oneactuation per nostril delivered volume of ~0.134 mL.

The α-adrenergic sympathomimetic decongestants (e.g. phenylephrine,oxymetazoline, napthoazoline) act on vascular smooth muscle to causeconstriction and thereby limit blood flow to the vessels of the nasalcavity. The reduced blood volume of the nasal cavity lowers resistanceto airflow and relieves nasal congestion. These nasal sprays come withthe warning of “do not use for more than three days” because of therisks of rebound hyperemia (rhinitis medicamentosa), when usage isstopped. In spite of this rebound danger, these “decongestant” solutionsare widely available. Here, we show that two DAPA compounds arecompatible with a commercial preparation of 1% phenylephrine HCl.Immediate cooling was observed when a DAPA compound was added to thephenylephrine HCl preparation. The results are shown in Table 10.

TABLE 10 Testing of Intranasal Decongestant with Cooling Agent Adjunct*Agent Decongestant Effects Vehicle** Phenylephrine No irritation ordiscomfort was reported HCl*** after use of the spray in four out offour subjects. There were also no sensations of coolness. DIPA-1-9Phenylephrine No discomfort was reported and no 5 mg/mL HCl***rhinorrhea was observed in the four tested subjects. Immediatesensations of cooling or cold were noted. DAPA-2-6 Phenylephrine Nodiscomfort was reported and no 5 mg/mL HCl*** rhinorrhea was observed inthe four tested subjects. Immediate sensations of cooling or cold werenoted. Tests were conducted in 4 normal volunteers without rhinitis.Subjects sprayed with one actuation three times a day for 3 consecutivedays. **The vehicle was distilled water. DAPA-2-5, DAPA-2-6 was added toExtra Strength Sinus Relief, CVS Health, containing phenylephrinehydrochloride 1.0%. The DAPA compounds are completely soluble in thissolution at the tested concentrations of 5 mg/mL. ***One actuation pernostril delivered volume of ~0.078 mL.

Study 6 Efficacy of Combinations in Adherence/Compliance and inTreatment of Rhinitis

From the previous experiments, it was concluded that DAPA compounds canbe combined with standard intranasal medications to obtain a coolingeffect with rapid, immediate onset and a sense of open breathing.Further studies were conducted on allergic rhinitis patients usingcombinations with nasal sprays containing triamcinolone acetonide(XinYang Company) and azelastine HCl (Meda Pharmaceuticals). The goalswere to determine adherence/compliance and efficacy of thesecombinations.

The measurement of adherence/compliance was the “weight of medicationsconsumed (WMC)”, that is, the weight of the spray bottle before andafter a 5 day period of use [Loh et al. A clinical survey on compliancein the treatment of rhinitis using nasal steroids, Eur. J. Allergy Clin.Immunol. 59, 1168-1172 (2004)]. It was found that the WMC was greater by33% in the triamcinolone group and by 40% in the azelastine group if thecooling agent was present. The main reason given by the subjects formore frequent use of the DAPA combination was because the subjects likedthe immediate drug effect and hence were less likely to forget to spray.The TNSS was also improved in the group that received the cooling agentcombination: by 28% in the triamcinolone group and by 45% in theazelastine group. The triamcinolone group objected to the “soapy” feelof the steroid spray and thereby used it less often. By contrast, theazelastine group felt immediate relief and was enthusiastic in adherencewithout, surprisingly, any complaints of bitter tastes.

In four of the six subjects in the cooling agent/azelastine group, therewas also the opinion that the “allergic rhinitis” condition hadpermanently been reduced in severity. These individuals continued to usethe combination beyond the 5-day trial period on an as needed basis. Theresults suggested the combination may have a disease-modifying effect onallergic rhinitis in addition to immediate symptomatic relief of itch,sneezing, rhinorrhea, and a feeling of nasal congestion. Some of thesecases are described further in the examples.

Case Study 1.

A 70-year old male subject had long-standing seasonal allergic rhinitissince he first noticed nasal symptoms on a golf course in spring, 45years ago. The likely trigger was grass pollen because the allergymanifested itself most often after rainstorms followed by periods of dryweather. Over the years, he learned to control nasal congestion andrhinorrhea with oral antihistamines by taking a daily dose of 10 mg ofloratadine, supplemented during the hay fever season by two 60 mgfexofenadine tablets daily, and when necessary 5 mg of chlorpheniramine.He had tried an intranasal steroid spray [Flonase®], but found thedelivery method “messy” and leaving objectionable sensations in the noseand mouth which interfered with gustation.

This season his allergy was heralded by the onset of severe bouts ofviolent sneezing, about 10 to 15 sneezes in a 15 min period, followed byrhinorrhea and congestion. He volunteered to try the DAPA compounds ofthis discovery and stopped using oral antihistamines. He started with aDIPA-1-9 2 mg/mL swab and right away noticed the disappearance ofsneezing and rhinorrhea which lasted for at least 12 hr. He found that asingle daily DIPA-1-9 1 mL swab was sufficient to block all symptoms ofhay fever. No odor, irritation, or taste was detected from the swab. Theindividual had a sense of free and unobstructed airflow in the nasalcavity. He no longer used any oral antihistamines.

This individual then volunteered to repeat the experience with otheranalogs with some of the results as shown in Table 5. He noted thecooling effects of 2-6 and 2-7 on the nostril skin, and some initialitching and rhinorrhea with these compounds. But he was also sure thatthese analogs were effective in preventing the symptoms of hay fever. Hetried swabs with DIPA-1-8 at a higher concentration of 4 mg/mL andwaited for hay fever symptoms to recur. To his surprise, symptoms didnot recur until after 5 days. He said it was as if the drug had curedthe disease. His wife also noted that he had stopped snoring andsnorting during sleep when he was using the swabs. Swabbing with justdistilled water was not effective in controlling his hay fever symptoms.He pronounced his cure as being “miraculous” because the rhinorrheadisappeared, although he still had an occasional sneeze. The only sideeffect he noted from the DAPA compounds was occasional stuffinessbecause of crusted and dried mucus on his nasal membranes. This problemwas easily solved by rinsing his nose with tap water.

Case Study 2.

A 50-year old male subject is a distinguished scientist at aworld-renowned institute of research in physiology. He has a MD and aPhD degree. The subject suffers from perennial rhinitis of many years.He stated that the rhinorrhea is “always there” and seeing specialistsand taking standard medications such as intranasal steroids andantihistamines were minimally effective to help control the symptoms. Henoted that on average his rhinorrhea can be estimated by the 10 Kleenextissues he deposits each day into his waste paper basket! He volunteeredto try swabs containing DIPA-1-9, 2 mg/mL and was given instructions onhow to apply the solution onto the anterior nasal vestibule. He reportsthat “The results are truly amazing. For the first time in many years, Iwoke up in the morning without any rhinorrhea.” He noted that he nowuses zero or only one or two Kleenex tissues for his nose each day. Heremarked on the “strong drying effect on the mucous membranes of thenasal cavity after repeated use” and suggested that there might be aninhibitory drug effect on serous gland secretion in the nasal mucosa.When asked if he felt coolness or cold when applying DIPA-1-9 2 mg/mLinto his nostril, he said “neither term is correct; the sensation is ofcomfortable freshness.” He was of the opinion that a slightly higherconcentration, e.g. 3 or 4 mg/mL of DIPA-1-8 or DIPA-1-9 instead of 2mg/mL might be more effective in gaining full control of his rhinorrhea.

A 65-year old male is retired and lives in Las Vegas, in a gatedcommunity with a golf course. But during spring he suffers severely fromallergic rhinitis and allergic conjunctivitis. The conjunctivitis isespecially annoying because he likes to play in professional pokertournaments. He volunteered to try the DIPA-1-9 2 mg/mL swabs and notedthat it worked well for his rhinitis. Surprisingly, he also found thathis pruritic conjunctivitis was relieved. Upon closer questioning, itwas clear that he did not apply the swabs to his eyes but had usedgenerous amounts in his nasal cavity. He had been instructed to squeezehis nostrils gently to disperse the liquid after applying the swab tothe anterior nasal vestibule. Apparently, he had applied too much liquidand squeezed the applied droplets so that there was retrograde flow upthe nasal-lachrymal duct so that his eyelids received the DIPA-1-9formulation. He remarked that his eyelids felt cool and comfortable andthe itchiness in his eyes was gone.

A 55-year old male subject manifested severe and persistent nasalcongestion. He was diagnosed as having chronic rhinosinusitis andendoscopic sinus surgery was recommended, but he was reluctant toundergo this procedure. He blamed his condition on not taking good careof his allergic rhinitis during his youth. All attempts at previousmedical therapy were not successful. He tried the isotonic saline nosedrops containing 2 mg/mL of DAPA-1-9 and pronounced the clearing effectas a “miracle”. He said that now he could sleep well at night. He usedthe nose drops on an as needed basis for two weeks, but decided that hewas not “cured” and agreed to try a combination of DAPA-1-9/azelastinehydrochloride combination. After two weeks of use, he said that hisnasal congestion had diminished to the point where he only needed to usethe nose drops on an occasional basis. He said the drug effect qualifiedas a “disease-modifying effect.”

Case Study 3

A 45-year old female subject was a professional tennis player. She hadnasal congestion from the common cold before an important match andasked if she could try the swab. She was given a swab containing 4 mg/mLof 2-6. She said the swab was fully effective in reducing her congestionand she was very pleased that she won her match. Subsequently, she wentto Hong Kong to visit her grandfather. It was in February and she notedthe air pollution was severe, and going out into the streets gave herthe sniffles. She had been given swabs containing DIPA-1-9 2 mg/mL andshe said use of these swabs reduced the irritant effects of breathingthe polluted air.

The efficacy of the swabs in controlling the symptoms of nasalstuffiness from the common cold was confirmed in two other subjects. Theeffects, however, were not as dramatic as with hay fever symptoms. Onesubject remarked that when “The nose was completely stuffed up, it wasdifficult to inhale and self-administer the contents of the swab.” It ismore likely that the common cold virus causes a wider area ofinflammation on the turbinate mucosa, and hence the medication needs tohave a wider distribution to abrogate the sense of stuffiness.Nevertheless, there was a beneficial effect on the subjective symptomsof congestion from the common cold.

Case Study 4

A 45-year old male subject with severe seasonal rhinitis volunteered totest various substances by intranasal swab delivery. He had previouslytried the Zicam Nasal Swabs for “gentle Allergy Relief” but noted thatthe gel on the swabs felt a little bit “slippery” and uncomfortable whenapplied to the nasal cavity. He said that the instructions andinformation on the Zicam box was “use 1 tube every 4 hours” and “optimalresults may not be seen for 1 to 2 weeks.” He said that the gelcongealed after opening in less than 24 hr so it was not possible to usethe same swab for multiple applications.

He tried the DIPA-1-9 2 mg/mL swab with and without the addition ofCPS-030 5 mg/mL. He noted that the CPS-030 added swab had a faster onsetof cooling action, but may have also increased his rhinorrhea.Monomenthyl glutarate and Cooler 10 added to DIPA-1-9 at 8 mg/mL did notenhance efficacy when tested in this subject. The DIPA-1-9 swab,however, effectively controlled his symptoms, especially the sneezing,with an immediate onset of effect. He only had to use the same swab andapply it twice a day to gain complete control of his symptoms. Thissubject remarked that icilin powder, inhaled from the radial fossa, wasstill the best medication for him because one dose was sufficient for 24hr control of his symptoms. He recommended that the icilin powder beadded to the DIPA-1-9 swab. Overall, he was very pleased with theefficacy of the DIPA-1-9 swab.

Case Study 5

A 49-year female subject went for a month vacation to the South ofFrance in March when the mimosa and cypress trees were in full bloom anddeveloped severe symptoms of allergic rhinitis. She had vigorous boutsof sneezing and profuse nasal secretions, and used up box after box ofKleenex tissues. Oral antihistamines such loratadine, cetirizine,chlorpheniramine, and fexofenadine were not effective, in part becauseshe did not adhere to a carefully regulated dosing regimen. She tried aintranasal steroid spray but complained that it gave her a messy“greasy” feeling in her nasal secretions. After using nose dropscontaining DAPA-1-9 in isotonic saline and a nasal spray of DAPA-1-9combined with azelastine HCl, her symptoms were controlled within 24 hrand she was able to resume her normal activities. She remarked that thecooling sensation and immediate relief of nasal congestion from thecombination product enabled her to follow a structured self-dosingschedule.

In summary, a class of DAPA compounds with cooling effects have beenidentified that can be combined with standard intranasal medicationsthat are used to treat the nasal discomforts associated with rhinitis.These molecules are water soluble and compatible current topicalintranasal medications such as intranasal steroids and intranasalantihistamine. The combinations are used to enhance the efficacy of themedications and to improve patient adherence to a dosage regimen.

REFERENCES

A number of publications are cited herein in order to more fullydescribe and disclose the discovery and the state of the art to whichthe discovery pertains. Each of these publications is incorporatedherein by reference in its entirety into the present disclosure.

1. A composition useful for relieving sensory discomfort in a nasalcavity, comprising: a 1-[Dialkyl-phosphinoyl]-alkane of Formula 1(O═)PR₁R₂R₃ wherein each of R₁, R₂, is either isopropyl or sec-butyl andR₃ is a linear alkyl group of 6 to 9 carbons, said1-[Dialkyl-phosphinoyl]-alkane being carried as a therapeuticallyeffective amount in a liquid solution; and, an intranasal steroid or anintranasal antihistamine.
 2. The composition as in claim 1 formulated ina dosage form suitable for intranasal administration.
 3. The compositionas in claim 2 wherein the dosage form is adapted for use as a nasalspray, as nasal drops, or as an irrigation solution.
 4. The compositionas in claim 1 wherein the 1-[Dialkyl-phosphinoyl]-alkane is present inthe solution at a concentration of 1 to 10 mg/mL.
 5. The composition asin claim 1 wherein the 1-[Dialkyl-phosphinoyl]-alkane is selected fromthe group consisting of 1-[Diisopropyl-phosphinoyl]-octane,1-[Diisopropyl-phosphinoyl]-nonane, 1-[Di-sec-butyl-phosphinoyl]-hexaneand 1-[Di-sec-butyl-phosphinoyl]-heptane.
 6. The composition as in claim1 wherein the intranasal steroid is selected from the group consistingof triamcinolone acetonide, beclomethasone dipropionate, budesonide,fluticasone propionate, fluticasone furoate, ciclesonide, and mometasonefuroate.
 7. The composition as in claim 1 wherein the intranasal steroidis triamcinolone acetonide.
 8. The composition as in claim 1 wherein theintranasal antihistamine is azelastine hydrochloride.
 9. The compositionas in claim 1 wherein the 1-[Dialkyl-phosphinoyl]-alkane is1-[Diisopropyl-phosphinoyl]-nonane which is present in the solution as aconcentration of 1 to 10 mg/mL.
 10. The composition as in claim 9wherein the dosage form is adapted for use as a nasal spray or as nasaldrops.
 11. The composition as in claim 1 wherein the sensory discomfortis rhinitis.
 12. The composition as in claim 1 wherein the sensorydiscomfort is allergic or perennial rhinitis, rhinitis of the commoncold, or rhinosinusitis.
 13. A method for relieving sensory discomfortdue to rhinitis, comprising: intranasally administering atherapeutically effective amount of a composition comprising a1-[Dialkyl-phosphinoyl]-alkane and an intranasal steroid or anintranasal antihistamine, the 1-[Dialkyl-phosphinoyl]-alkane being ofFormula 1(O═)PR₁R₂R₃ wherein each of R₁, R₂, is either isopropyl or sec-butyl andR₃ is a linear alkyl group of 6 to 9 carbons, said1-[Dialkyl-phosphinoyl]-alkane being carried as a therapeuticallyeffective amount in a liquid solution.
 14. The method as in claim 13wherein the rhinitis is caused by allergic or perennial allergicrhinitis.
 15. The method as in claim 13 wherein the composition is inthe form of an aerosol, nasal drops, a nasal spray or an irrigationsolution.
 16. The method as in claim 13 wherein the1-[Dialkyl-phosphinoyl]-alkane is selected from the group consisting of1-[Diisopropyl-phosphinoyl]-octane, 1-[Diisopropyl-phosphinoyl]-nonane,1-[Di-sec-butyl-phosphinoyl]-hexane and1-[Di-sec-butyl-phosphinoyl]-heptane.
 17. The method as in claim 13wherein the intranasal steroid is selected from the group consisting oftriamcinolone acetonide, beclomethasone dipropionate, budesonide,fluticasone propionate, fluticasone furoate, ciclesonide, and mometasonefuroate.
 18. The method as in claim 13 wherein the intranasal steroid istriamcinolone acetonide.
 19. The method as in claim 13 wherein theintranasal antihistamine is azelastine hydrochloride.
 20. The method asin claim 15 wherein the intranasal irrigation solution is an isotonic orhypertonic saline solution.
 21. The method as in claim 13 wherein theadministering is to a patient and increases compliance or adherence touse of the intranasal steroid or the intranasal antihistamine by thepatient.
 22. A method of relieving sensory discomfort in a nasal cavity,comprising: providing an aqueous liquid composition having a therapeuticamount of 1-[Diisopropyl-phosphinoyl]-nonane therein and an intranasalmedication selected from a group of an intranasal steroid, an intranasalantihistamine, a sympathomimetic decongestant, a mast cell stabilizer,an antimuscarinic agent, a hypertonic saline solution, or an isotonicsaline solution; and, instructing a user in intranasally administeringsaid composition.
 23. The method as in claim 22 wherein theadministering is by means of a nasal spray as nasal drops, or as anirrigation solution.
 24. The method as in claim 22 wherein theintranasal steroid is selected from the group consisting oftriamcinolone acetonide, beclomethasone dipropionate, budesonide,fluticasone propionate, fluticasone furoate, ciclesonide, and mometasonefuroate.
 25. The method as in claim 22 wherein the intranasal steroid istriamcinolone acetonide.
 26. The method as in claim 22 wherein theintranasal antihistamine is azelastine hydrochloride.
 27. The method asin claim 22 wherein the therapeutic amount of1-[Diisopropyl-phosphinoyl]-nonane is a concentration of 1 to 10 mg/mL.28. The method as in claim 22 wherein the sensory discomfort isrhinitis.
 29. The method as in claim 22 wherein the rhinitis is allergicor perennial rhinitis.
 30. The method as in claim 22 wherein theadministering is to a patient and increases compliance or adherence touse of the intranasal steroid or the intranasal antihistamine by thepatient.